Interactions between circadian clocks and feeding

Interactions between circadian clocks and feeding behaviour.

Satish Kumar Sen

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UNIVERSITE DE STRASBOURG France UNIVERSITE D'AMSTERDAM PAYS-BAS ÉCOLE DOCTORALE Sciences de la Vie et de la Santé Institut des Neurosciences Cellulaires et Intégratives de Strasbourg

THÈSE EN COTUTELLE

présentée par :

SATISH KUMAR SEN Soutenue le : 09 July 2018

Pour obtenir le grade de : Docteur de l’université de Strasbourg & Docteur de l'université d'Amsterdam Discipline/Spécialité :

Sciences du vivant / Neurosciences

Interactions between circadian clocks and feeding behaviour

THÈSE dirigée par: Dr. CHALLET E. Prof. KALSBEEK A.

Docteur, Université de Strasbourg Professeur, Université d'Amsterdam

RAPPORTEURS: Prof. OSTER H. Prof. SCHLICHTER R. Dr. FELDER-SCHMITTBUHL M.P. Prof. LA FLEUR S. Dr. YI C.X.

Professeur, Université de Lübeck Professeur, Université de Strasbourg Docteur, Université de Strasbourg Professeur, Université d'Amsterdam Docteur, Université d'Amsterdam

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Interactions between circadian clocks and feeding behaviour ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam

op gezag van de Rector Magnificus prof. dr. ir. K.I.J. Maex

ten overstaan van een door het college voor promoties ingestelde commissie in het openbaar te verdedigen in het Institut des Neurosciences Cellulaires et Intégratives de Strasbourg op maandag 9 Juli 2018, te 14:00 uur

door

SATISH KUMAR SEN geboren te Bina, India

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PROMOTIECOMMISSIE: Promotores :

Overige leden :

Prof. Dr. A. Kalsbeek

Universiteit van Amsterdam

Dr. E. Challet

Universiteit van Straatsburg

Prof. Dr. H. Oster

Universiteit van Lübeck

Prof. Dr. R. Schlichter

Universiteit van Straatsburg

Dr. M.P. Felder-Schmittbuhl

Universiteit van Straatsburg

Prof. Dr. S.E. la Fleur

Universiteit van Amsterdam

Dr. C.X. Yi

Universiteit van Amsterdam

Faculteit der Geneeskunde

Dit proefschrift is tot stand gekomen in het kader van het NeuroTime programma, een Erasmus Mundus Joint Doctorate, met als doel het behalen van een gezamenlijk doctoraat. Het proefschrift is voorbereid in: het Nederlands Herseninstituut en in het Academisch Medisch Centrum (AMC), Faculteit der Geneeskunde, van de Universiteit van Amsterdam; en in het Institut des Neurosciences Cellulaires et Intégratives van de Université de Strasbourg. This thesis has been written within the framework of the NeuroTime program, an Erasmus Mundus Joint Doctorate, with the purpose of obtaining a joint doctorate degree. The thesis was prepared in: the Netherlands Institute for Neuroscience (NIN) and in the Academic Medical Centre (AMC), Faculty of Medicine at the University of Amsterdam; and in the Institut des Neurosciences Cellulaires et Intégratives of the Université de Strasbourg.

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Contents Chapter 1 ........................................................................................................................... 9 General introduction Chapter 2 ......................................................................................................................... 63 Ultradian feeding in mice not only affects the peripheral clock in the liver, but also the master clock in the brain .................................................................................................. 63 Chapter 3 ......................................................................................................................... 99 An ultradian feeding schedule in rats differentially affects peripheral clocks in liver, brown adipose tissue and skeletal muscle and lipid metabolism, but not the central clock in SCN ............................................................................................................................... 99 Chapter 4 ....................................................................................................................... 137 Differential effects of diet composition and timing of feeding behavior on rat Brown adipose tissue and skeletal muscle peripheral clocks. .................................................... 137 Chapter 5 ....................................................................................................................... 167 Expression of the clock gene Rev-erbα in the brain controls the circadian organization of food intake and locomotor activity, but not daily variations of energy metabolism ...... 167 Chapter 6 ....................................................................................................................... 195 Discussion and perspectives ........................................................................................... 195 Summary ......................................................................................................................... 209 Samenvatting .................................................................................................................. 216 Résumé............................................................................................................................ 220 PhD Portfolio .................................................................................................................. 231

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Chapter 1 General Introduction

Chapter 1 ............................................................................................................................. 9 General Introduction ........................................................................................................... 9 1.

Introduction of rhythms ......................................................................................... 10 1.1. Circadian rhythms .......................................................................................................... 11 1.2. Infradian rhythms ........................................................................................................... 12 1.3. Ultradian rhythms ........................................................................................................... 13

2. 3.

Molecular mechanism underlying generation of circadian rhythms ..................... 13 Circadian clocks: A multi-oscillatory system ........................................................ 15 3.1 Master clock in the suprachiasmatic nucleus (SCN) ................................................. 15 3.2 Peripheral clocks ............................................................................................................. 21

4. 5.

Circadian control of plasma metabolites and hormones ........................................ 24 Interactions between the circadian clock system, feeding and metabolism. ......... 30 5.1 Restricted feeding and calorie restriction .............................................................. 30 5.2 Diet and its impact on circadian clocks. ..................................................................... 33 5.3 Clock genes in relation to metabolic genes ................................................................. 34 5.4 The nuclear receptor REV-ERBα in relation to circadian clock and metabolism .............................................................................................................................. 36

6. Aim of my thesis ........................................................................................................... 41 Chapter 2 Effects of ultradian feeding on central and peripheral clocks in mice ......................................................................................................................................... 41 Chapter 3 Effects of ultradian feeding on central and peripheral clocks in rats ........... 42 Chapter 4 Differential effects of diet composition and timing of feeding behaviour on rat brown adipose tissue and skeletal muscle peripheral clocks .............. 42 Chapter 5 Role of the clock gene Rev-erbα in feeding and energy metabolism ............................................................................................................................. 42

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1. Introduction of rhythms The sun and moon are important for the existence of life on earth. The sun gives energy in the form of light, the moon reflects light from the sun and makes tidal waves. Life on our terrestrial planet originated nearly about 3.8 billion years ago from the transition of nonliving to living systems through the process of biogenesis. The moon orbits around the earth and the earth moves around the sun. The rotation of the earth around its own axis gives rise to day and night cycles, according to which the physiology of most living organisms responds. Most living organisms, whether they are uni- or multi-cellular, express day and night cycles. Daily rhythms were first described in a written report by Androsthenes of Thasos around 4th century B.C.E. (mentioned in (Bretzl, 1903) after observing the daily periodic movement of the leaves of the tamarind tree, Tamarindus indica. In 1729, the French astronomer Jean-Jacques d’Ortous de Mairan observed that every day the leaves of the Mimosa pudica opened during the day and closed at night. To confirm whether this opening and closing of the leaves was due to sunrise and sunset, he kept a plant in constant darkness and observed that the leaves still opened and closed at the usual time of day. Through this experiment he demonstrated the existence of an internal timing system in Mimosa pudica. Thirty years later, Henri-Louis Duhamel du Moceau, a French botanist, showed that the movement of leaves in constant darkness was independent of the environmental temperature changes, thus providing further evidence for the internal origin of this rhythm (McClung, 2006). In the 1930’s Erwin Bünning, a German biologist, evidenced the genetic origin of circadian rhythms by crossing bean plants with different endogenous periods. In the daily life of animal species, daily rhythms are expressed in a wide range of biological processes, such as their rest-activity cycle, hormone release and body temperature rhythm. Daily rhythms can be defined as sequences of events having a defined period of 24 hours and specific phase and amplitude for that particular event (Figure 1). In the human physiological system many events are organized in time, with most (locomotor) activity occurring during daytime and sleep and rest during the night. Also other events take place at specific times of day, like body temperature reaching its nadir level at the beginning of the night and melatonin being released from the pineal gland only during the night.

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Figure 1: Parameters used to describe a biological rhythm: The period is defined as the time required to complete one cycle. Mesor is the average value around which the variable measured oscillates. The amplitude of the rhythms is defined as the difference between the mesor and the acrophase (or bathyphase). Acrophase and bathyphase represent the time points when the parameter measured shows the highest or the lowest values, respectively.

1.1. Circadian rhythms The daily rhythmicity of the external behavior of an organism is due to its internal circadian clock (Pittendrigh, 1993). The term “circadian”, which was first used by Franz Halberg in 1950s, arises from the Latin words circa which means “around” and dies for “day”. The circadian rhythms comprise biological processes with a period length of about 24 h (20-28 h), thus completing their cycle in approximately one day. Circadian rhythms are endogenous in nature. These self-sustained circadian rhythms can be investigated by measuring external behavioural and physiological parameters like locomotor activity and body temperature under constant (lightening) conditions. In 1960, Jürgen Aschoff observed lengthening and shortening of period length, in respectively nocturnal and diurnal animals, under constant light conditions, in response to an increase in light intensity. When there are no external environmental cues such as light, i.e., under constant dark (DD) conditions, with food ad libitum, circadian rhythms in rats and mice persist and show so-called free-running (Figure 2) (Aschoff, 1965). A similar observation was made in humans, i.e., the existence of free-running rhythms in constant conditions, by Michel Siffre, a French explorer and scientist, staying voluntarily in an isolated cave for two months with no external time cues (Siffre, 1963). A process known as entrainment allows 11

circadian rhythms to be synchronized to the external environment by setting the endogenous period to exactly 24 h. Hence circadian entrainment is important for living organisms to be in phase with the daily variations in the environment. The light-dark (LD) cycle is the primary external entraining cue for the synchronization of circadian rhythms. All mammalian species respond to LD cycles by timing their body physiology and metabolism to a specific phase of the LD cycle, like eating during the dark phase and sleeping during the light phase in nocturnal species.

Figure 2: Schematic representation of rodent single plotted actogram. The above activity plot, or so-called actogram, represents a rhythm of locomotor activity initially entrained to the 24-h light-dark (LD) cycle. Upon transfer to constant darkness (DD), a free-running circadian rhythm resumes with its endogenous period.

The circadian clock drives the daily rhythm in body temperature, but also the sleep-wake cycle and level of motor activity affect this rhythm (Tokura and Aschoff, 1983; Refinetti and Menaker, 1992). The rhythm in body temperature is due to the difference in circadian variation of heat loss and production (Aschoff, 1983). Likewise, there are daily variations in many hormones, such as leptin from white adipose tissue, insulin from the endocrine pancreas and melatonin from the pineal gland. 1.2. Infradian rhythms Rhythms with a period (much) longer than 24 h are known as “infradian rhythms". These rhythms include 4-5 day rhythms to monthly rhythms, such as the estrous cycle in rodents and the menstrual cycle in humans, respectively. This category also includes lunar 12

rhythms, and seasonal rhythms like the shedding of leaves, reproduction in seasonal animals and migration of birds, among many others. 1.3. Ultradian rhythms Rhythms that occur with a period much shorter than 24 h are called “ultradian rhythms” (Halberg and Reinberg, 1967). These rhythms are known to be associated with feeding behaviour and various physiological processes, like pulsatile hormonal secretion, cardiovascular function, like heartbeat and blood pressure, and respiratory exchange of gases.

2. Molecular mechanism underlying generation of circadian rhythms In the early 1970s Seymour Benzer and his student Ron Konopka were the first to identify the mutation affecting circadian behaviour in Drosophila melanogaster in a gene they called Period (Konopka and Benzer, 1971). Later in the 1980s the molecular clock research work in Drosophila melanogaster was continued by the scientists Jeffrey Hall, Michael Rosbash and Michael Young who were honoured the Nobel prize for medicine or physiology in 2017 for deciphering the principles of the molecular mechanism of the circadian clock, i.e., the transcriptional-translational feedback loop (TTFL). The identification of clock genes in Drosophila helped in cloning mammalian clock genes. The molecular machinery of the circadian clock involves a collection of clock genes which are expressed rhythmically and control the clock oscillations. The complete core clock mechanism relies on positive and negative transcriptional, post-transcriptional, translational and post-translational feedback loops (Shearman et al., 2000; Reppert and Weaver, 2001) (Figure 3). In mammals, the two key elements involved in the molecular clock machinery are the basic helix-loop-helix (bHLH)/PAS-containing transcription factors BMAL1 and CLOCK (King et al., 1997; Gekakis et al., 1998; Hogenesch et al., 1998; Griffin et al., 1999; Bunger et al., 2000). BMAL1 and CLOCK heterodimerize and activate via E-box sequences the rhythmic transcription of other clock genes, like the Period genes (Per1, Per2 and Per3), the Cryptochrome genes (Cry1 and Cry2) (Griffin et al., 1999; Kume et al., 1999; van der Horst et al., 1999; Vitaterna et al., 1999; Shearman et al., 2000), Reverbα and Rorα (Preitner et al., 2002; Sato et al., 2004; Triqueneaux et al., 2004; Akashi and Takumi, 2005), and various clock-controlled genes like Vasopressin

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(Avp), D site albumin promoter binding protein (Dbp) (Jin et al., 1999; Ripperger et al., 2000) and many others. These transcripts are translated in the cytoplasm.

Figure 3: Molecular mechanism of mammalian circadian clock. Transcriptional-translational feedback loops of core clock genes. CLOCK and BMAL1 dimerize and activate the transcription of other clock genes such Per1&2, Cry1&2, Rorα, Rev-erbα by binding to E-boxes in their promoter region. After transcription of these clock genes they are translationally released in the cytoplasm. Translated PER and CRY proteins heterodimerize and translocate back to the nucleus to inhibit the transcription of their own genes by binding to BMAL1 and CLOCK. Clock proteins undergo post-translational modifications and ubiquitination for proteosomal degradation. Another loop involves the translocation of REV-ERBs and RORs into the nucleus to modulate transcription after binding to RORE sequences in the promoters of Bmal1 and Clock genes to activate or repress their transcription.

From the cytoplasm, some of the clock proteins are translocated back to the nucleus to regulate their expression by interacting with the CLOCK/BMAL1 complex and interfering with its transcriptional activity. These proteins also regulate themselves through another loop by binding to the RORE sequences in the promoter region of CLOCK and BMAL1. The circadian expression of CLOCK and BMAL1 is regulated both positively and negatively by RORs and REV-ERBs, respectively. Many clock proteins also undergo post-translational modifications. This is an important mechanism for regulating more precisely the phase, amplitude and period of the circadian clocks, by modulating the stability and turnover of various clock proteins (Bellet and Sassone-Corsi, 2010). Important post-translational modifications are phosphorylation, sumoylation, acetylation, and ubiquitination. Phosphorylation regulates the cellular 14

localization and stability of various clock proteins and maintains the circadian period close to 24 h (Lee et al., 2001). Phosphorylation occurs at the phosphor-acceptor site on its substrate by kinases. Casine kinase Iε (CKIε) is one of the kinases which phosphorylate the PER proteins. A mutation in the gene encoding for CKIε, characterized as tau mutation, shortens the period of circadian rhythmicity in the Syrian hamster (Lowrey et al., 2000). Phosphorylation can also cause the recruitment of ubiquitin ligase adapter Fbox protein bTrC and target the clock proteins for ubiquitination-mediated proteasomal degradation (Eide et al., 2005; Shirogane et al., 2005; Bellet and Sassone-Corsi, 2010). There are various kinases which phosphorylate other clock proteins, including CKIε (Eide et al., 2002), mitogen-activated protein kinases (MAPKs) (Sanada et al., 2002), and CK2α (Tamaru et al., 2009) and glycogen synthase kinase 3β (GSK3 β) that all phosphorylate BMAL1 (Sahar et al., 2010). Furthermore, GSK3 β on its turn phosphorylates other clock proteins, such as CRY2 (Harada et al., 2005), PER2 (Iitaka et al., 2005), REV-ERBα (Yin et al., 2006) and CLOCK (Spengler et al., 2009). In addition to phosphorylation, sumoylation also controls the turnover of the clock proteins. The small ubiquitin-related modifier 1 (SUMO 1) protein, which regulates the process of SUMOylation in clock proteins (Cardone et al., 2005), sumoylates BMAL1 at a conserved lysine (K259) residue present in the PAS domain linker. Mutation of genes encoding for ubiquitin ligases can also abolish circadian rhythmicity. The F-box-type E3 ligase FBXL3 ubiquitinates the CRY1/2 proteins (Busino et al., 2007; Godinho et al., 2007; Siepka et al., 2007). Ovine CRY1 degradation can be reduced by FBXL3 and its homologue FBXL21 (Dardente et al., 2008). REV-ERBα undergoes degradation by the E3 ligases HUWE1/ARF-BP1 and PAM/MYC-BP2 and also by lithium which is an inhibitor of GSK3 β (Yin et al., 2010; Stojkovic et al., 2014). Another E3 ligase UBE3A binds and destabilizes BMAL1 (Gossan et al., 2014). Moreover, another pathway through which REV-ERBα is targeted for ubiquitination involves degradation by F-box protein FBXW7, resulting in an increased circadian amplitude (Zhao et al., 2016a).

3. Circadian clocks: A multi-oscillatory system 3.1 Master clock in the suprachiasmatic nucleus (SCN) The lesion studies by Richter in 1967 provided the evidence for the involvement of the anterior hypothalamus in the regulation of daily rhythms of locomotor activity. In 1972

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Moore and Lenn identified that the SCN (Figure 4) in the anterior hypothalamus receive direct input from the retina.

Figure 4: Localisation of the suprachiasmatic nucleus (SCN) in mice. Coronal section of a mouse brain showing the localization of the SCN above the OC. The below cresyl violet staining shows the cellular density in the SCN. OC: optic chiasma; 3V: third ventricle. Adapted from the Franklin-Paxinos atlas (Paxinos, 2004).

Later that same year, Stephan and Zucker observed that bilateral electrolytic lesions of the SCN resulted in loss of daily rhythms of drinking behaviour and locomotor activity. Moreover, in the same year, Moore and Eichler found also that the daily rhythm in the adrenal amount of corticosterone was abolished in SCN-ablated rats (Moore and Eichler, 1972; Stephan and Zucker, 1972) (Figure 5). Not only behavioural responses, but also physiological responses, like body temperature, turned out to be SCN dependent. Lesions of the SCN eliminated the circadian rhythm of body temperature, although a few studies suggested a weak but significant circadian rhythmicity in body temperature after SCN lesions (Dunn et al., 1977; Fuller et al., 1981; Satinoff and Prosser, 1988), which may have been due to incomplete SCN lesions. In vivo electrical recordings in rats displayed the rhythmic firing rate of SCN neurons (Inouye and Kawamura, 1979) and in vitro electrical recording of cultured neonatal rat SCN cells provided the evidence for an endogenous pacemaker and single-cell circadian oscillators (Welsh et al., 1995). These

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findings indicate that this circadian clock in the SCN is composed of multiple single-cell oscillators (Liu et al., 1997).

Figure 5: Abolition of locomotor activity and drinking rhythms by SCN lesions in rats. Simultaneously recorded activity (A) and drinking (D) rhythms from an un-operated control rat (No. 226, top) and a rat with a bilateral suprachiasmatic lesion (No.9, bottom) during a 50-day period about 2 months after surgery. The control rat’s drinking activity is entrained to the light-dark cycle. There is no entrainment or circadian periodicity in the record of the rat with the lesion. Adapted from (Stephan and Zucker, 1972).

The SCN was finally accepted as the central or master clock after the transplantation of fetal SCN tissue in SCN-lesioned animals and restoration of daily behavioural rhythms of locomotor activity (Drucker-Colin et al., 1984; Lehman et al., 1987; DeCoursey and Buggy, 1989; Ralph et al., 1990; LeSauter et al., 1996; Silver et al., 1996), although these transplants did not restore neuroendocrine rhythms (Meyer-Bernstein et al., 1999). Daily rhythms in body temperature, rest-activity and hormone release are controlled via neuroendocrine and autonomic nervous output pathways of the SCN (LeSauter et al., 1996; Ueyama et al., 1999; Kalsbeek et al., 2011). Light or photic cues are detected and integrated by the retina, and conveyed from the retina to the ventrolateral region of the SCN via the retinohypothalamic tract (Hendrickson et al., 1972; Moore and Lenn, 1972; Moore and Card, 1985; Moore, 1995). A small subpopulation of the retinal ganglion cells are intrinsically photosensitive because they contain the photopigment melanopsin. Many of these melanopsin-containing ganglion cells innervate the SCN (Provencio et al., 2000; Gooley et al., 2001; Hannibal et al., 2002; Hattar et al., 2002). Anterograde and retrograde tracing studies revealed the detailed topography of the SCN efferents (Watts and Swanson, 1987; Watts et al., 1987; Kalsbeek et al., 1993). The central SCN clock sends direct projections to secondary clocks of the 17

hypothalamus in the paraventricular nuclei (PVN), the ventromedial hypothalamic nuclei (VMH), the dorsomedial hypothalamic nuclei (DMH), the arcuate nuclei (ARC) and the retrochiasmatic area, whose daily timing is thus synchronized to the SCN clock. In addition, the SCN also sends its efferents directly to a few extra-hypothalamic areas such as the paraventricular nucleus of the thalamus (PVT), habenula and amygdala (AMY) (Kalsbeek and Buijs, 2002; Saper et al., 2005; Dibner et al., 2010) (Figure 6).

Figure 6: Efferent pathways from the SCN SCN projections (red) to hypothalamic (yellow), thalamic (green) and sub-cortical (pink) brain regions. AMY, amygdala; ARC, arcuate nucleus; BNST, bed nucleus of the stria terminalis; DMH, dorsomedial hypothalamic nucleus; 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. Modified from (Dibner et al., 2010).

In turn, several hypothalamic nuclei, including the ARC and DMH, convey feeding and metabolic signals to the SCN clock (Challet and Mendoza, 2010). The ARC and DMH are involved in the regulation of feeding and energy metabolism (Guilding and Piggins, 2007; Williams and Elmquist, 2012). The ARC contains two populations of neurons which behave opposite with regard to their control of feeding behaviour. One population of neurons synthesizes Neuropeptide Y (NPY) and Agouti-related peptide (AgRP), which are both orexigenic. The other group of neurons synthesizes Pro-opiomelanocortin (POMC) and Cocaine and amphetamine regulated transcript (CART), which are both anorexigenic (Akabayashi et al., 1994; Steiner et al., 1994; Xu et al., 1999). Loss or destruction of leptin-sensitive and NPY-sensitive neurons in the ARC nucleus results in disturbed daily rhythms in food intake (Wiater et al., 2011; Li et al., 2012). Like the arcuate nucleus, DMH neurons are also sensitive to feeding-related hormones such as leptin (Elmquist et al., 1997). The DMH has been suggested to play a critical role in the regulation of a wide 18

range of behavioural rhythms. Excitotoxic lesions of the DMH disrupt circadian rhythms of wakefulness, feeding, locomotor activity and plasma corticosterone. Additionally, the DMH connects with several hypothalamic nuclei including the lateral hypothalamic area and PVN. The afferent neurons of DMH innervating the ventrolateral preoptic nucleus are largely GABAergic, while those innervating the lateral hypothalamic area are mainly glutamatergic (Chou et al., 2003; Saper et al., 2005). The DMH also appears to be involved in food entrainment (Mistlberger, 2006). During restricted feeding the DMH exhibits a robust oscillation of mPer expression and this oscillation persist for a few days after the food deprivation (Mieda et al., 2006). Thus the DMH could be an important area that mediates the effects of SCN output on several behavioural and physiological rhythms and may play a role in daily rhythm of feeding/fasting. Anatomy and cell types of the SCN. Anatomically and functionally, the SCN contains at least two major subdivisions: a ventral "core" region and a dorsal "shell" region based on afferent and efferent projections and neuropeptide expression (Ibata et al., 1989; Antle and Silver, 2005; Gamble et al., 2007; Kiss et al., 2008). On the one hand, the ventral core of the SCN expresses Gastrinreleasing peptide (GRP) and Vasoactive intestinal polypeptide (VIP) (Abrahamson and Moore, 2001; Antle and Silver, 2005). GRP levels in the rat SCN reach peak levels during the resting phase and gradually decrease during the dark phase, while VIP remains low during the light phase and gradually reaches peak levels during the dark period (Shinohara et al., 1993). On the other hand, the dorsal shell contains mainly neurons that express Arginine vasopressin (AVP) along with calretinin (CAR) (Moore et al., 2002) (Figure 7).

Figure 7: Distribution of principal neuropeptides in the SCN SCN neurons expressing the neuropeptides VIP, GRP, AVP and the neurotransmitter GABA. Many of the SCN neurons within the dorsal shell express AVP and GABA. The SCN neurons in the ventral core contain VIP and GRP. AVP: arginine vasopressin; GABA: Gamma amino-butyric acid; VIP: vasoactive intestinal polypeptide; GRP: gastrin-releasing peptide.

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mRNA expression of Avp is highest during daytime under light-dark conditions, in both nocturnal and diurnal rodents, and this rhythm persists in constant darkness (Dardente et al., 2004). Moreover, also the rhythmic release of AVP is higher during the daytime (Kalsbeek et al., 1998). Additionally, SCN neurons also contain many other neuropeptides (Cheng et al., 2002), such as for instance prokineticin 2 (PK2), a multifunctional secretory protein (Zhou and Cheng, 2005; Negri et al., 2007), which plays a role in transmission of the circadian signals from the SCN (Zhou and Cheng, 2005; Zhang et al., 2009). Photic entrainment of SCN clock In order to maintain its daily rhythmicity synchronized with the outside world, the SCN clock requires a synchronizing signal with a 24-h period, for instance the environmental light-dark (LD) cycle. In the absence of such a synchronizing input, the SCN clock starts to free-run with a period different from, although still close to 24 h. Photic entrainment of the SCN clock occurs via retinal ganglion cells which project to the SCN through the retinohypothalamic tract (RHT) (Panda et al., 2002; Foster et al., 2007; Panda, 2007) and is dependent on the timing of the light exposure. In response to photic stimulation, RHT terminals release glutamate and pituitary adenylate cyclase activating peptide (PACAP) in the SCN, which stimulate their receptors on SCN neurons and cause the transcription of the clock genes Per1 and Per2 (Albrecht et al., 1997; Shearman et al., 1997; Shigeyoshi et al., 1997; Reppert and Weaver, 2002).

Figure 8: Photic input signal transduction pathways in the SCN neuron. Solid and dashed lines indicate the direct and indirect phase-shifting pathways, respectively. BIT, brain immunoglobulin-like molecules with tyrosine-based activation motifs; CaMKII, calcium/calmodulin kinase II; CRE, cAMP response element; CREB, CRE-binding protein; PACAP, pituitary adenylate cyclaseactivating peptide; PKGII, cGMP-dependent protein kinase II. Adapted from (Hirota and Fukada, 2004).

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In most of the nocturnal and diurnal animals, exposure to light pulses during the early night causes a phase delay, while a light pulse in the late night results in a phase advance. By contrast, exposure to a light pulse around midday does not have any resetting effect on the master clock. Light exposure activates a series of pathways with induction of early and immediate responses, such as transcription of c-fos and phosphorylation of ERK (Morris et al., 1998; Obrietan et al., 1998).Light-induced phase delays are associated with upregulated expression of Per1 and Per2 in the SCN core and later with Per2 expression in the SCN shell (Hamada et al., 2001; Yan and Okamura, 2002; Hamada et al., 2004). By contrast, light-induced phase advances are associated only with increased expression of Per1 in the core SCN, but not Per2 (Antle and Silver, 2005) (Figure 8). 3.2 Peripheral clocks Each cell and organ have their own clock. These clocks are entrained by the master clock in the SCN and remain synchronized under regular laboratory conditions (i.e., stable LD cycle, food and water ad libitum, constant ambient temperature). The master clock sends its output signals to almost each peripheral tissue of the body via neuroendocrine and autonomic nervous pathways. The entrainment of peripheral clocks by the master clock, the light-dark cycle, feeding-fasting cycle, and diet composition had been studied extensively (Figure 9).

Figure 9: Organization of circadian timing system. The master clock, located in the suprachiasmatic nuclei (SCN) of the hypothalamus, adjusts the timing of many secondary clocks/oscillations in the brain and peripheral organs, in part via nervous pathways (dotted red lines). Light perceived by the retina is the potent synchronizer of the SCN clock (dashed yellow arrow), while meal time can synchronize peripheral clocks (blue arrows). Modified from (Delezie and Challet, 2011).

Liver clock The liver is an important organ in the control of energy homeostasis. In particular, the liver is an important organ for glucose uptake, storage, and production. Hepatic glycogen 21

anabolism and catabolism show daily rhythms (Kida et al., 1980; Roesler et al., 1985; Roesler and Khandelwal, 1985). Many genes encoding enzymes involved in hepatic glucose metabolism display a circadian expression pattern (Lamia et al., 2008). Some of these metabolic genes lose their rhythmicity in mice with a liver-specific knock-out of the Bmal1 gene, while others remain rhythmic, probably through systemic cues. Glucose transporter (Glut2) in the liver is critical for exporting glucose from the liver. Its maximal expression is found during fasting, while during the feeding phase its expression is lower, then limiting glucose exported from the liver (Schmutz et al., 2012). Under an LD cycle temporal restricted feeding can shift the phase of clock gene expression in the liver, for up to 12h. These changes in the liver clock are much faster than any other peripheral clocks, such as kidney, pancreas and heart (Damiola et al., 2000; Stokkan et al., 2001). The changes in phase and amplitude of rhythmic genes in the liver involve feeding/fasting cues (Atger et al., 2017). Entrainment of the liver clock induced by a restricted feeding cycle is independent of the SCN clock and the light-dark cycle (Hara et al., 2001; Stokkan et al., 2001; Bae and Androulakis, 2017), but does involve temperature cues as shown with experiments using dampened temperature cycles (Damiola et al., 2000; Brown et al., 2002). Feeding/fasting cycles trigger the secretion of various hormones, many metabolites and affect the intracellular redox state by changing the NADH/NAD+ ratio. The hepatic clock is also altered by the amount of food and the interval between feeding time-points, but it remains unaffected by the frequency of feeding as long as the interval remains fixed (Kuroda et al., 2012). Skeletal muscle clock Nearly 45% of the body mass is composed of skeletal muscle (Goodpaster et al., 2000; Hoppeler and Fluck, 2002), making skeletal muscle one of the largest tissues of the body. Skeletal muscle is strongly implicated in the maintenance of glucose homeostasis as it takes up 80% of postprandial glucose (DeFronzo et al., 1981b; Ferrannini et al., 1988). In skeletal muscle, over 2300 genes involved in myogenesis, transcription and metabolism, are expressed rhythmically (McCarthy et al., 2007; Pizarro et al., 2013; Harfmann et al., 2015). Muscle physiology is entrained either directly by the molecular clock (Yamazaki et al., 2000) or indirectly by other rhythmic factors such as feeding time, neuro-humoral signals, and locomotor activity that are controlled by the SCN clock. The communication of the SCN molecular clock to the skeletal muscle is mediated through neuro-humoral and 22

temperature signals (Balsalobre et al., 2000; Brown et al., 2002; Abraham et al., 2010; Saini et al., 2012). The muscle clock gets desynchronized from the SCN clock when external stimuli, such as feeding and exercise, are out of phase with the regular LD cycle (Mayeuf-Louchart et al., 2015). Feeding/fasting cycles can synchronize the muscle clock, as shown by the restricted feeding schedule (Damiola et al., 2000; Opperhuizen et al., 2016). In mice, fasting for 24h does not disturb the muscle clock (Dudek and Meng, 2014). In addition to the feeding/fasting cycle, also activity cues, such as scheduled physical exercise, may act as a Zeitgeber for the muscle clock (Yamazaki et al., 2000; Yamanaka et al., 2008; Wolff and Esser, 2012). Disruption of circadian rhythms in skeletal muscle results in impaired glucose tolerance and insulin sensitivity (Yoo et al., 2004; Harfmann et al., 2015). Restricted feeding and scheduled activity in PER2:LUC mice results in a shift in gene expression of the muscle molecular clock (Wolff and Esser, 2012). Restricted feeding limiting food access to the light/resting phase desynchronizes the skeletal muscle clock from the liver clock of rats, as clock genes in muscle lose their rhythmicity when rats are fed with a chow diet during the resting phase (Reznick et al., 2013; Opperhuizen et al., 2016), but remain rhythmic with an unchanged phase when fed a high-fat diet during the resting phase (Reznick et al., 2013). Brown adipose tissue clock Like the liver and skeletal muscle, brown adipose tissue (BAT) is another metabolically active organ, but in this case specifically involved in non-shivering thermogenesis. BAT is highly enriched in capillaries that supply oxygen and lipid as a substrate to generate heat through its endogenous thermogenic process. BAT contains numerous small-sized lipid droplets, as well as iron-rich mitochondria expressing uncoupling protein-1 (UCP1, thermogenin). Mice deficient in UCP1 (Enerback et al., 1997) and BAT (Lowell et al., 1993) helped to demonstrate the functionality of UCP1 and BAT for the generation of heat through non-shivering thermogenesis (Matthias et al., 2000; Golozoubova et al., 2001). BAT is also a major organ for glucose uptake (Cawthorne, 1989), through glucose transporters GLUT1 and GLUT4, which are activated by cold exposure and noradrenergic signalling (Nikami et al., 1992; Dallner et al., 2006; Bartelt et al., 2011). Due to its plasma lipids and plasma glucose lowering and insulin sensitivity increasing effect, increased BAT activity helps in reducing metabolic disorders related to obesity and diabetes 23

(Nedergaard et al., 2011). Various genetic models of clock gene mutants provided evidence for the involvement of the BAT clock in thermogenesis (Chappuis et al., 2013; Gerhart-Hines et al., 2013; Nam et al., 2016). The activation of BAT by various highcalorie diets, such as high fat or high sugar diets, is likely through increased UCP1 levels, thereby providing a potential mechanism to limit weight/fat gain (Rothwell and Stock, 1979; Bukowiecki et al., 1983; Mercer and Trayhurn, 1987; Moriya, 1994; LeBlanc and Labrie, 1997).

4. Circadian control of plasma metabolites and hormones Daily variations in plasma hormones and metabolites are under the control of the circadian timing system, but also affected by the feeding-fasting and rest-activity cycles. Dysregulation of these plasma metabolites may result in various metabolic disorders such obesity, dyslipidemia, type 2 diabetes, and hypertension. In this thesis we concentrated especially on the following metabolites and hormones: glucose, free fatty acids, corticosterone, insulin, leptin, and melatonin (Figure 10). Glucose metabolism Glucose, the major energy reservoir of the cell, is required for normal functioning. Body uptake of glucose occurs mostly from carbohydrate-rich diets via the systemic circulation. The liver stores glucose in the form of glycogen. Plasma glucose concentrations show a daily rhythm, as reported both in animals and humans. The daily rhythm of basal glucose concentrations is SCN-dependent and gets abolished after bilateral lesions of SCN, while the rhythm still persists when rats are fasted or fed a 6-meals-a-day feeding schedule (Nagai et al., 1994; Kalsbeek et al., 1998; La Fleur et al., 1999). The plasma glucose concentration depends on influx of glucose from gut and liver and efflux of glucose mostly to brain, muscle and adipose tissues (Kalsbeek et al., 2006). Glucagon produced by pancreatic α-cells acts on the liver for stimulating the synthesis of hepatic glucose through the process of glycogenolysis and gluconeogenesis (Pilkis and Granner, 1992; Kurukulasuriya et al., 2003). In rats, the daily rhythm of plasma glucose concentrations peaks prior to the onset of activity (La Fleur et al., 1999; Challet et al., 2004; Cailotto et al., 2005b). The stimulation of sympathetic fibers that innervate the liver increases glucose production through glycogen phosphorylase activation (Shimazu and Fukuda, 1965), while activation of the parasympathetic pathway to the liver decreases hepatic glucose production through an inhibitory action on glycogen synthase (Shimazu, 1967). Hepatic 24

denervation studies in rats provided the evidence for a critical role of the SCN, mediated through the autonomic nervous system, in the daily rhythm of plasma glucose concentrations (Kalsbeek et al., 2004; Cailotto et al., 2005b). Apart from the SCN, this pathway involves other hypothalamic nuclei such as the PVN. Injections of a GABA-A antagonist or an NMDA agonist in the vicinity of the PVN resulted in activation of PVN neurons and caused hyperglycemia independent of insulin and corticosterone release (Kalsbeek et al., 2004), but possibly involving increased release of glucagon.

Figure10: Schematic representation of the circadian timing system in a nocturnal rodent. The suprachiasmatic nuclei (SCN), site of the master clock, are mostly reset by light cues (in yellow) perceived by the retina. Secondary clocks in the brain and peripheral tissue (only a few are shown) are phase controlled in part by temporal cues from the master clock, via the autonomic nervous system (blue arrows). Peripheral glands release rhythmically hormones. Brain-controlled feeding/fasting, sleep/wake cycles and changes in body temperature (not shown) are also modulators of peripheral rhythmicity. WAT, white adipose tissue. Modified from (Challet, 2015).

The circadian clock and clock components also play an important role in the regulation of glucose metabolism at other levels. Various clock gene mutants presented a number of metabolic disorders, including hyperglycemia, dyslipidemia, hepatic steatosis and reduced gluconeogenesis. More specifically, mutation of the Clock gene has a major impact on glucose metabolism, such as reduced gluconeogenesis and increased insulin sensitivity, hyperglycemia, decreased glucose tolerance and dampened oscillations of hepatic glycogen and glycogen synthase 2 (Rudic et al., 2004; Turek et al., 2005; Kennaway et al., 2007; Doi et al., 2010; Marcheva et al., 2010). Similarly, knock-out of Bmal1 leads to altered glucose metabolism. Global and liver-specific Bmal1 knockout mice develop glucose intolerance (Lamia et al., 2008). A pancreas-specific knockout of Bmal1 also induces impaired glucose tolerance, as well as hypoinsulinemia (Marcheva et al., 2010). 25

Also mutations in other clock genes such as Per2, Cry1/Cry2, and Rev-erbα impact on glucose metabolism with differential effects on glycemia, glycogen storage, and glucose tolerance (Schmutz et al., 2010; Delezie et al., 2012; Zhao et al., 2012; Zani et al., 2013). Lipid metabolism The liver also plays a pivotal role in lipid metabolism. It is the major site for converting carbohydrates into fatty acids and triglycerides which are than exported and stored in adipose tissue. Free fatty acids are derived from the circulation, as well as from de novo synthesis from acetyl Co-A or malonyl-CoA. The free fatty acids are converted into triglycerides (TGs) in hepatocytes which are than further used for the production of VLDL particles for export (Bradbury, 2006). Lipids are the major source of stored energy in the white adipose tissues (WAT) of mammals. When energy requirements of the body cannot be full-filled by circulating energy metabolites such as carbohydrates, a breakdown of lipids occurs through the process of lipolysis from WAT. Lipolysis involves hydrolysis of TGs into glycerol and free fatty acids via activation of the hormone sensitive lipase. Plasma free fatty acids show a daily rhythm which is dependent on the SCN (Yamamoto et al., 1984, 1987; Dallman et al., 1999). Plasma apolipoproteins help in the transportation of other lipids such as TG and cholesterol (Pan and Hussain, 2009; Challet, 2013). Intestinal production of lipoproteins may cause a rise in plasma TGs and cholesterol in nocturnal rodents during their active phase (Pan and Hussain, 2007). Diurnal variations of plasma lipids in mice are under circadian control, and are altered under constant lightening and restricted feeding conditions (Pan and Hussain, 2007). Most of the genes in the intestine such apolipoprotein B, apolipoprotein AIV, intestinal triglycerides transport protein and intestinal fatty acid binding protein show diurnal variations during lipid uptake and metabolism (Pan and Hussain, 2007, 2009; Pan et al., 2010). Mutations or circadian disruptions of the molecular clock machinery have a pronounced influence on lipid rhythms. Clock mutant mice lose the day-night rhythmicity in the absorption of macronutrients due to loss of rhythm in the intestinal absorption (Pan and Hussain, 2009). Similarly, clock mutant mice also express altered circadian rhythmicity of the genes involved in TG synthesis and lipolysis ((Kudo et al., 2007; Tsai et al., 2010; Shostak et al., 2013) and are characterized by hypertriglyceridemia (Turek et al., 2005). Pparα is well known for its involvement in the regulation of lipid metabolism. Bmal1 knock-out mice display down26

regulation of Pparα in liver suggesting a close connection between Pparα and Bmal1 (Canaple et al., 2006). Knock-down of Bmal 1 in 3T3-L1 adipocytes results in decreased adipocyte differentiation and lipogenesis gene expression, while Bmal1 knock out mice have high levels of circulating fatty acids resulting in an unusual accumulation of fat in liver and muscle (Shimba et al., 2011). Embryonic fibroblast cells of the Bmal 1 knockout mice failed to differentiate into adipocytes (Shimba et al., 2005). Daily oscillations of plasma TGs are disrupted in the Bmal1 knock-out mice (Rudic et al., 2004; Bunger et al., 2005). Per2 knock-out mice have altered lipid metabolism (Grimaldi et al., 2010) et al. 2010). Liver lipidomic analysis of Per1/2 null mice fed under ad libitum or night time restricted feeding still shows oscillation in TGs in an anti-phasic manner suggesting oscillation of TGs in the absence of a functional clock. Night time restricted feeding reduces hepatic triglycerides levels in wild-type mice (Adamovich et al., 2014). Like the other clock genes, also Rev-erbα plays an important role in lipid metabolism, more precisely in adipogenesis (Fontaine et al., 2003; Duez and Staels, 2008b; Delezie et al., 2012). In addition, it also regulates TGs and TG rich lipoprotein metabolism (Raspe et al., 2002). It has been shown in rats that Rev-erbα represses various apolipoproteins A-I which are the major protein constituents of high-density lipoproteins (HDL) (Vu-Dac et al., 1998). Rev-erbα-deficient mice display high levels of hepatic apoC-III expression, plasma TGs and TG-rich very low density lipoproteins (VLDL) (Raspe et al., 2001; Raspe et al., 2002). Under regular chow-feeding conditions these mice display increased adiposity, and a period of 24 h fasting increases more fatty acid mobilization in the knockout mice as compared to wild-type littermates. When fed with a high-fat diet, the Rev-erbα knockout mice are more prone to metabolic disturbances and lipogenic factors are more activated compared to wild-type animals (Delezie et al., 2012). Corticosterone rhythm Plasma corticosterone levels peak prior to the onset of activity, which is just before lights off in nocturnal animals (Cheifetz, 1971; Ixart et al., 1977; Carnes et al., 1989). The SCN clock in the hypothalamus regulates the adrenal production and secretion of glucocorticoids. The circadian regulation of glucocorticoid release is mediated via the hypothalamic-pituitary-adrenal axis and the autonomic nervous system. AVP, one of the principal neuropeptides of the SCN projections towards the PVN/DMH area, presents a diurnal release in the cerebrospinal fluid and in the PVN/DMH and SCN region (Reppert 27

et al., 1981; Kalsbeek et al., 1995; Buijs et al., 1999). Micro-infusion of AVP in PVN and DMH inhibits corticosterone release (Kalsbeek et al., 1992), while infusion of an AVP antagonist in PVN and DMH at the time of highest AVP release has a stimulatory effect on corticosterone release (Kalsbeek et al., 1992; Kalsbeek et al., 1996a). The circadian release of corticosterone in both nocturnal (rats) and diurnal (Arvicanthis) animals are in phase with the onset of daily activity. The administration of AVP in the PVN of diurnal animals stimulates corticosterone release, that is, it has opposite effects to those in nocturnal animals (Kalsbeek et al., 2008). The feeding-fasting cycle also strongly influences the activity of the hypothalamicpituitary-adrenal axis. In mice and rats, daytime restricted feeding provokes a bimodal pattern of corticosterone secretion, the first peak corresponding to feeding time while the second peak occurs at dusk at a similar phase to ad libitum feeding conditions (Le Minh et al., 2001). However, the first, feeding driven peak is independent of the SCN (Krieger et al., 1977). The circadian rhythm of circulating glucocorticoids is thought to synchronize a number of peripheral clocks (Dickmeis, 2009). Melatonin rhythm In mammals, the pineal gland secretes melatonin, a lipophilic hormone, only in the night. The rhythmic release of melatonin is under control of the SCN, but is also highly influenced by the presence or absence of light (Pevet and Challet, 2011). In the absence of light, melatonin is synthesized during the subjective night phase, while in the presence of light, either during the regular day or at night, melatonin synthesis is inhibited. The secretion of melatonin from the pineal gland always takes place at night in both nocturnal and diurnal animals. Therefore, it has been considered as a phase marker of the SCN clock (Cajochen et al., 2003; Arendt and Skene, 2005). Kalsbeek et al. demonstrated that the daily rhythm of melatonin synthesis is caused by a rhythmic alternation of glutamatergic and GABAergic outputs from the SCN (Perreau-Lenz et al., 2004). These glutamatergic and GABAergic signals control the activity of pre-autonomic PVN neurons that are in control of the sympathetic inputs to the pineal gland. During the light period GABAergic neurons in the SCN provide inhibitory signals onto these pre-autonomic neurons in the PVN, whereas in the dark period glutamatergic inputs from the SCN stimulate these preautonomic neurons to start the secretion of pineal melatonin (Kalsbeek and Fliers, 2013).

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Plasma melatonin also provides feedback signals to the SCN clock (Pevet and Challet, 2011). In rodents, melatonin has phase resetting properties on SCN oscillations (Armstrong and Redman, 1985). Restricted feeding combined with calorie restriction in rats causes a small but significant phase advance of the daily rhythm of pineal melatonin (Challet et al., 1997a). In SCN-lesioned rats, restricted feeding restores the rhythmic transcription of the rate limiting enzyme arylalkylamine-N-acetyltransferase, possibly via sympathetic fibers (Feillet et al., 2008a). Melatonin is known to influence other hormone rhythms such as leptin, corticosterone and insulin (Peschke and Peschke, 1998; Gunduz, 2002; Alonso-Vale et al., 2008; Chakir et al., 2015). Leptin rhythm Leptin is a hormone which is secreted from adipocytes in the white adipose tissue and plays a major role in the regulation of food intake and energy homeostasis by exerting its effect on hypothalamic neurons. Leptin acts on the NPY and POMC neurons in the arcuate nucleus by binding to the LEPR-B. More precisely, leptin inhibits the orexigenic NPY/AgRP neurons, while it activates the anorexigenic POMC- and CART-expressing neurons as a result of which food intake is decreased (Schwartz et al., 2000; Kalra and Kalra, 2003; Sobrino Crespo et al., 2014). Circulating leptin concentrations are related to fat mass and adiposity in humans and rodents (Ahima et al., 1996; Considine and Caro, 1996; Havel et al., 1996; Kolaczynski et al., 1996; Elimam and Marcus, 2002). During food restriction, leptin levels decrease, leading to increased appetite and reduced energy expenditure (Velkoska et al., 2003). Leptin signals probably also modulate peripheral clocks as indicated by the alteration of clock gene expression in liver and adipose tissue of leptin-deficient (ob/ob) and leptin-resistant (KK-Ay) mice and Zucker rats (Motosugi et al., 2011). Plasma leptin levels in mice and rats show a diurnal rhythm with a nocturnal peak. This rhythm is sex dependent (i.e., with much higher amplitude in females as compared to male mice) and is abolished under 24-fasting conditions (Ahren, 2000). Complete destruction of the SCN in rats results in a loss of the diurnal rhythmicity, while a 6-meals-a-day feeding schedule has no major effect on its rhythmicity. Furthermore, the rhythm of plasma leptin is controlled by the SCN via the sympathetic fibers innervating WAT (Kalsbeek et al., 2001). Rhythmic secretion of leptin is also modulated by the circadian clock within the adipocytes (Otway et al., 2009). Plasma leptin may give feedback to the SCN as SCN cells express leptin receptors and an in vitro study in rats showed leptin-induced phase advances of the SCN clock (Prosser and Bergeron, 2003). 29

Hence leptin may play an important role in connecting circadian clocks and energy metabolism. Insulin rhythm Alike to the hormones cited above, insulin also shows daily variations in plasma concentrations, which are under control of the SCN (la Fleur et al., 2001; Rudic et al., 2004; Shi et al., 2013). Of note, insulin sensitivity also displays daily rhythmicity (la Fleur et al., 2001). Insulin, which is secreted by pancreatic β-cells in response to a meal, regulates blood glucose by favouring the entry of glucose into metabolically active tissues, thus reducing glycemia (Patton and Mistlberger, 2013). The secretion of insulin after a meal results in acute changes in Per2 and Rev-erbα expression in the liver (Tahara et al., 2011; Yamajuku et al., 2012). Mice with a global deletion of the clock genes Cry1 and Cry2 present hyperinsulinemia (Barclay et al., 2013), whereas specific ablation of the clock genes Clock and Bmal1 in the pancreas results in hypoinsulinemia (Marcheva et al., 2010; Sadacca et al., 2011). The 6-meals-a-day feeding schedule nicely demonstrates the stimulatory effect of the SCN on insulin release during night time meals (Kalsbeek et al., 1998). SCN-lesioned mice lose their daily insulin rhythm and display hyperinsulinemia (Coomans et al., 2013). In diabetic rats, the phase of the circadian clock in the heart is shifted, suggesting a role for hyperglycaemia and/or altered insulin signalling on the cardiac clock (Young et al., 2002).

5. Interactions between the circadian clock system, feeding and metabolism. 5.1 Restricted feeding and calorie restriction Limiting food access in rodents to either the active or resting phase, with no caloric restriction, is known as restricted feeding. Rodents under restricted feeding conditions adjust and adapt to their new feeding-fasting schedule within a few days (Honma et al., 1983; Froy et al., 2006). Feeding restricted every day for a single, short period of time entrains various food-entrainable oscillators (FEO) and synchronizes the physiological and behavioural rhythms to the feeding opportunity, for instance, a period of increased locomotor activity prior to every day access to food. This robust increase in locomotor activity is known as food-anticipatory activity. Restricted feeding also leads to anticipatory increases in body temperature, several metabolic cues, heart rate and 30

secretion of glucocorticoids (Saito et al., 1976; Comperatore and Stephan, 1987; Mistlberger, 1994; Hara et al., 2001; Boulamery-Velly et al., 2005; Saper et al., 2005; Hirao et al., 2006). When rats are entrained to a restricted feeding opportunity during the middle of the resting phase, c-FOS protein expression in the DMH is shifted to the daytime, indicating that the timing of the activation of the DMH is linked to meal time (Angeles-Castellanos et al., 2004). The food-anticipatory activity persists in SCN-lesioned animals which indicates that the FEOs are located outside the master clock, likely in neural structures from the hypothalamus to brainstem that regulate feeding behaviour (Mistlberger and Antle, 2011). Restricting food access to the resting phase also inverses or shifts clock gene expression in peripheral tissues such as liver, lungs, and kidneys, but not in the SCN clock, thereby uncoupling central and peripheral clocks (Figure 11) (Damiola et al., 2000; Hara et al., 2001; Stokkan et al., 2001; Cassone and Stephan, 2002; Schibler et al., 2003; Hirota and Fukada, 2004). The restricted feeding paradigm also phase shifts the peak expression of the clock genes in several brain areas outside of the SCN, such as cerebral cortex and striatum, compared to animals fed ad libitum (Wakamatsu et al., 2001; Feillet et al., 2008a). A high fat diet in combination with the time restricted feeding prevents mice from developing metabolic disorder such as obesity, hyperinsulinemia, and hepatic steatosis if the high fat diet is provided during the usual active period (Mendoza et al., 2008a; Hatori et al., 2012).

Figure 11: Daytime feeding changes the phase of clock gene expression in the liver but not in the suprachiasmatic nucleus (SCN). (A) Circadian accumulation of Per1 and Per2 mRNA levels in liver. (B) Circadian accumulation of Per1 and Per2 in SCN. Adapted from (Damiola et al., 2000).

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Figure 12: Immunoreactive expression of AVP and PER2 in the SCN of caloric restricted (T-CR) and control (AL) mice. (A) AVP-ir nuclei and (B) PER2-ir nuclei in the SCN from mice under a 12-h LD cycle and AL (black symbols) or T-CR (White symbols) at different Zeitgeber times (ZT-0, lights on; ZT-12, lights off. (C) Schematic presentation showing that daily hypocaloric feeding affects the SCN clock. Black and White top bars indicate the LD cycle. Vertical arrows indicate the meal time. Adapted and modified from (Mendoza et al., 2007a).

When rats that are housed under a regular LD cycle are exposed to restricted feeding coupled with caloric restriction (hypocaloric diet), they display phase advances in their locomotor activity rhythm, body temperature cycle, and the daily rhythm of plasma melatonin (Challet et al., 1997a; Wakamatsu et al., 2001; Feillet et al., 2008a). Caloric restriction is elicited by reducing the total caloric intake to 60-70% of ad libitum values without malnutrition. Animals under caloric restriction fed either one or two daily meals or exposed to intermittent daily fasting displayed an increased lifespan (Masoro, 1995; Masoro et al., 1995; Froy and Miskin, 2010), and delayed onset of age-related diseases such as cancer, diabetes, kidney disease, and cataract (Weindruch et al., 1997; Roth et al., 2002; Koubova and Guarente, 2003; Roth et al., 2004; Masoro, 2005). Caloric restriction can entrain the SCN clock and modulate photic entrainment (Challet et al., 1997b; Challet et al., 2003; Mendoza et al., 2005; Resuehr and Olcese, 2005). Caloric restricted mice express a rise in body temperature in anticipation of the scheduled food access (Duffy et al., 1989). A hypocaloric meal provided during daytime also affects the temporal organization of the SCN clock in mice, with a shifted SCN clock and clock outputs (Figure 12) (Mendoza et al., 2005; Mendoza et al., 2007a). Rats on a six-meals-a-day feeding schedule coupled with hypocaloric meals also showed a phase advance of their locomotor activity and body temperature rhythms (Mendoza J et al. 2008). In conclusion, 32

caloric restriction affects the peripheral clock in liver similar to restricted feeding (Damiola et al., 2000; Hara et al., 2001; Stokkan et al., 2001), but contrary to timerestricted feeding it affects the central SCN clock as well (Mendoza et al., 2007a) via additional, as yet unknown mechanisms. 5.2 Diet and its impact on circadian clocks. Eating behavior is not only about “how much we eat”, but also about “what and when we eat”. The daily life style of humans nowadays is highly influenced by late evening activities, shift work, and high-caloric feeding. A high-caloric diet has a major impact on body physiology and metabolism, leading to obesity, diabetes, and other features of the metabolic syndrome. A high energy diet also leads to circadian disruption. Mice fed on a high-fat diet present disruption of behavioral rhythms with a dampened rhythm of locomotor activity and increased feeding duration together with alterations of circadian clock and metabolic gene expression in metabolically active tissues such as hypothalamus, liver, muscle and adipose tissue (Kohsaka et al., 2007; Barnea et al., 2009, 2010). High-fat feeding also affects the plasma levels of hormones involved in fuel utilization such leptin, insulin, corticosterone, prolactin, luteinizing hormone, thyroid stimulating hormone, testosterone and also pineal melatonin in rats, mice and humans (Havel et al., 1999; Cha et al., 2000; Cano et al., 2008; Honma et al., 2016). Mice on a high-fat diet display increased body mass index, and increased plasma metabolite concentrations such as glucose, and free fatty acids. High-fat feeding in mice also impairs circadian re-entrainment after a jetlag (Mendoza et al., 2008a). Furthermore, decreased light-induced phase shifts of mice fed with a high-fat diet correlate with a reduction of light-induced c-FOS and P-ERK in the SCN (Mendoza et al., 2008a). Mice fed with a high-fat, high-sugar diet (fcHFHS) in combination with short-term daytime feeding display desynchronized peripheral clocks, leading to the metabolic syndrome through leptin resistance, physical inactivity and fatty liver and adiposity (Yasumoto et al., 2016). Rats on fcHFHS for five weeks gain body weight over the control group, while those rats fed with either high-fat or high-sugar do not show an increase of body weight. The rise in body weight in the fcHFHS group is due to increased abdominal fat (la Fleur et al., 2010). Moreover, feeding behavior is modified, as characterized by increased meal numbers due to sugar intake without reducing meal size (la Fleur et al., 2007; la Fleur et al., 2014). Of note, rats fed on fcHFHS diet display no change in rhythms of body temperature or locomotor activity (la Fleur et al., 2007). 33

5.3 Clock genes in relation to metabolic genes Metabolic pathways are regulated by numerous metabolic genes involved in lipid and glucose metabolism and are tightly connected with the molecular mechanism of the circadian clock. The transcription factors peroxisome proliferator-activated receptors (PPARs) play an important role in linking metabolism to circadian clocks. PPAR expression is found to be rhythmic in mouse liver, skeletal muscle, white and brown adipose tissues (Lemberger et al., 1996b; Yang et al., 2006), which gives a clue of the tight interconnectivity between these two systems (Figure 11). PPARs belong to a superfamily of ligand-activated nuclear receptors. After binding to ligands such as free fatty acids and eicosanoids, PPARs heterodimerize with retinoid X receptors (RXRs). Then these complexes bind to PPAR responsive elements (PPRE) and activate the transcription of their target genes. There are three PPAR isoforms: PPARα, PPARβ/δ and PPARγ (Berger and Moller, 2002), each of them differs from each other by tissue-specific distribution, specificity toward particular ligands and functionality (Willson et al., 2000). PPARα, a metabolic sensor and lipid metabolizing gene in the liver, makes a direct link between the circadian clock and metabolism by binding a PPRE in the promoter of Bmal1 and regulating its expression positively (Lemberger et al., 1996a; Oishi et al., 2005; Canaple et al., 2006). PPARγ, the paralog of PPARα, is expressed highly in white and brown adipose tissues in which it regulates adipogenesis and lipid biosynthesis (Kliewer et al., 1997; Sheu et al., 2005; Medina-Gomez et al., 2007). Genetic ablation of PPARγ in mice is associated with behavioral changes by abolishing or dampening circadian rhythmicity, which affects body metabolism (Yang et al., 2012). It plays an important role in the regulation of heart rate and blood pressure by forming a feedback loop with BMAL1. Conditional knockout of PPARγ in the vascular system dampens the heart rate and blood pressure (Wang et al., 2008). PPARγ and its partner PPARα positively regulate expression of the clock gene Rev-erbα in the liver and PPARγ also promotes adipocyte differentiation (Gervois et al., 1999; Fontaine et al., 2003). The PPARγ coactivator 1α (PGC1α) is expressed rhythmically in mouse liver and muscle, thereby it is involved in connecting the molecular clock and energy metabolism. Its main function is the regulation of oxidative phosphorylation by mitochondrial biogenesis (Bellet and Sassone-Corsi, 2010). Genetic deletion of PGC-1α in mice leads to an abnormal locomotor activity pattern, disrupted body temperature rhythms, and disturbed energy metabolism (Lin et al., 2005; Feige and 34

Auwerx, 2007; Liu et al., 2007). PPARβ/δ is highly ubiquitous and expressed in most tissues of the body (Braissant et al., 1996). It has a role in the control of energy homeostasis (Coll et al., 2009; Asher and Schibler, 2011). A recent study demonstrated PPARβ/δ expression in the hamster SCN, and showed that a PPARβ/δ agonist amplifies phase delays of the locomotor activity rhythm in response to a light pulse (Challet et al., 2013). This possible direct link to the circadian clock needs to be investigated further. Sirtuin1 (SIRT1), a NAD+ dependent deacetylase, acts as a cellular nutrient sensor (Sahar and Corsi 2012). An increased NAD+/NADH ratio activates SIRT1 which links it to cellular energy metabolism (Bordone and Guarente, 2005). Previous reports showed that during caloric restriction SIRT1 activates PGC-1α via its deacetylation (Rodgers et al., 2005). SIRT1 is an important modulator of the circadian machinery (Asher et al., 2008; Nakahata et al., 2008). SIRT1 regulates circadian rhythms by deacetylation of histones at the promoter of clock genes, and non-histone proteins BMAL1 and PER2 display a circadian pattern of expression (Nakahata et al., 2009; Ramsey et al., 2009). This 24-h expression of NAD+ may be dependent on the rate limiting enzyme nicotinamide phosphoribosyl transferase (NAMPT), suggesting a close connection of the circadian clock and metabolic processes within the peripheral clock.

Figure 13: The mammalian circadian clock and its link to energy metabolism . Expression of Bmal1 and Rev‐erbα genes are controlled by PPARα and binding of RORs to RORE sequences. RORs need a co‐activator, PGC‐1α, which is phosphorylated by activated AMPK. In parallel, AMPK activation leads to an increase in NAD+ levels, which, in turn activate SIRT1. SIRT1 activation leads to PGC‐1α deacetylation and activation. Acetyl adenosine diphosphate ribose (Ac‐ADP‐r) and nicotinamide (NAM) are released after deacetylation by SIRT1. Adapted from (Froy and Miskin, 2010).

Energy metabolism also impacts the cellular redox status (Figure 13). A study by Rutter et al. showed that the cellular redox has an impact on the circadian clock such that the level 35

of pyridine nucleotides can modulate DNA binding of CLOCK-BMAL1 or Neuronal PAS Domain Protein 2 (NPAS2)-BMAL1 heterodimers (Rutter et al., 2001). Daily rhythms in the cellular redox state are observed in the liver by daily changes in the rate-limiting enzyme in the NAD+ salvage pathways and circadian-dependent regulation of the nicotinamide phosphoribosyl transferase (NAMPT) (Nakahata et al., 2009; Ramsey et al., 2009). The NAD+ levels in cells Poly (ADP-ribose) polymerase 1 (PARP-1) binds to the CLOCK-BMAL1 heterodimer and poly-ADP-ribosylates CLOCK during the early light phase. Knock-out of PARP-1 has been shown to affect the clock machinery in the liver in response to a change in feeding time and impair the food entrainment of peripheral circadian clocks. Daytime restricted feeding shifts the expression of clock genes and the auto-ADP-ribosylation of PARP-1 in liver. Hence feeding regulates the circadian expression of the PARP-1, and may thus via the poly-ADP-ribosylation of CLOCK also affect the molecular clock (Asher et al., 2010). Adenosine monophosphate (AMP) activates the protein kinase AMPK, thereby AMPK activity provides information about the cellular energy state via the AMP to ATP ratio (Davies et al., 1992). In particular, AMPK phosphorylates and destabilizes one of the core clock proteins, CRY1. In this way, AMPK sends timing signals about the cellular energy state directly to the molecular clock via CRY1 (Lamia et al., 2009). 5.4 The nuclear receptor REV-ERBα in relation to circadian clock and metabolism The nuclear receptor (NR) REV-ERBα is also known as NR1D1. REV-ERBα is a transcriptional repressor, encoded by the reverse strand of the thyroid hormone receptor cErbα, as well as its isoform which was discovered later on as REV-ERBβ (Lazar et al., 1989; Dumas et al., 1994; Forman et al., 1994). Until 2007 REV-ERBα was considered as an orphan nuclear receptor. Then, heme was discovered as the physiological ligand, which regulates the activity of REV-ERBα (Yin et al., 2007; Meng et al., 2008). Unlike other NRs, REV-ERBα lacks the carboxy-terminal activation function 2 (AF2) region in its ligand binding domain (LBD) (Dumas et al., 1994; Forman et al., 1994). The AF2 region identifies co-activators required for the transcription. Therefore, due to the lack of an AF2 region, REV-ERBα cannot activate transcription. It acts as a transcriptional repressor due to the binding of co-repressors such as the nuclear receptor co-repressor (NCoR) in the hydrophobic region (Renaud et al., 2000; Yin and Lazar, 2005). REV-ERBα binds to its response element called Rev-erbα response element (RRE, or RORE) containing six base 36

pair core motifs (A/G) GGTCA flanked by an A/T rich 5’ (Solt et al., 2012). REV-ERBα can also repress its own transcription via a RevDR2 binding site in its promotor (Adelmant et al., 1996). REV-ERBα binds either as a monomer or as a homodimer to the RevDR2/ROREs elements which consist of direct repeats of the core motif separated by two nucleotides (Harding and Lazar, 1993; Dumas et al., 1994; Retnakaran et al., 1994; Harding and Lazar, 1995). The binding competitor of REV-ERBα is Retinoic Acid Receptor-Related Orphan receptor α (RORα). Both transcription factors share the same DNA binding site, ROR response elements (RREs or ROREs), but behave in an opposite manner, REV-ERBα acting as a transcriptional repressor while RORα is a transcriptional activator (Duez and Staels, 2008b; Zhao et al., 2014). REV-ERBα members of NR family have diverse roles in different biological processes, such as circadian system, sleep regulation, reproduction, development, inflammation and energy metabolism. They also participate in various metabolic pathways, such as gluconeogenesis, adipocyte differentiation, bile acid synthesis, heme, cholesterol homeostasis and thermogenesis (Yin et al., 2007; Duez and Staels, 2008b; Le Martelot et al., 2009; Delezie et al., 2012; Nam et al., 2016). REV-ERBα was the first NR shown as a link between cellular metabolism and the circadian clock by acting as a circadian transcriptional repressor that regulates the expression of core clock genes and increases the robustness of clock oscillation, besides its involvement in intracellular metabolic pathways. REV-ERBα can also be considered as a clock-controlled gene, because it somehow mediates output pathways of the molecular clock in the SCN and peripheral tissues (Lazar et al., 1989; Balsalobre et al., 1998; Torra et al., 2000). The promoter sequence of Bmal1, a key positive limb element of core clock, contains RORE sequences to which REV-ERBα binds to inhibit its transcription (Preitner et al., 2002; Bugge et al., 2012; Cho et al., 2012). Mice with a knock-out for Rev-erbα show elevated expression of Bmal1, highlighting the repressive effect of REV-ERBα (Preitner et al., 2002). The transcription of Npas2 and Clock is also under the control of REV-ERBα because both of these genes contain RORE sequences in their promoter (Crumbley et al., 2010; Crumbley and Burris, 2011). An in vitro study showed that the E-box binding sites of BMAL1/CLOCK are present in the Rev-erbα promoter, thus suggesting a bidirectional transcriptional

regulation

of

Rev-erbα

though

BMAL1/CLOCK

transactivation

(Triqueneaux et al., 2004). These studies collectively indicate the transcriptional relationship between BMAL1/CLOCK and REV-ERB/ROR. REV-ERBα binds to the 37

corepressor N-CoR to repress transcription (Harding and Lazar, 1995; Hu and Lazar, 1999; Ishizuka and Lazar, 2003). The N-CoR/REV-ERBα complex interacts with multiprotein complex Histone deacetylase 3 (HDAC3) which deacetylates histone, causes chromatin compaction and represses Bmal1 transcription (Guenther et al., 2000; Guenther et al., 2001; Ishizuka and Lazar, 2003; Yin and Lazar, 2005). Glycogen synthase kinase 3β (GSK3β) phosphorylates and stabilizes REV-ERBα, while its stability can be modified with lithium (Yin et al., 2006). The cyclin-dependent kinase 1 (CDK1) phosphorylates REV-ERBα at its T275 site, and recognizes/recruits F-box protein, FBXW7α, for proteasome degradation, suggesting that the amplitude of rhythmic expression of REVERBα is dependent on CDK1-FBXW7 axis (Zhao et al., 2016a). 5.4.1 Role of REV-ERBα in behavioural responses. Global Rev-erbα knock-out mice show a shorter period length of locomotor activity (-0.5 h) compared to wild-type mice under constant light (LL) or constant dark (DD) conditions. Global Rev-erbα knock-out mice exposed to light pulses of 2 h in the late night display larger phase advances in locomotor activity rhythm (Preitner et al., 2002). Brainspecific Rev-erbα knock-out mice kept under a regular light dark cycle show a drastic reduction in the day-night amplitude of general locomotor activity. Furthermore, general locomotor activity is highly disturbed, showing either arrhythmicity or heterogeneity among individual free-running periods under DD condition. (Delezie et al., 2016). Food entrainment activity is impaired in global Rev-erbα knock-out mice with decreased anticipatory bouts of locomotor activity and body temperature whether animals are under light-dark or DD conditions, whereas brain deletion of Rev-erbα prevents foodanticipatory behaviour and thermogenesis (Delezie et al., 2016). Mice with global deletion of Rev-erbα with chow available ad libitum do not differ in food intake, general locomotor activity, and body temperature as compared to wild-type mice, while their respiratory quotient rhythm is altered during both day and night. Besides its role in feeding and metabolism, REV-ERBα also plays an important role in vascular inflammation (Sato et al., 2014b), sleep homeostasis, sleep-wake cycle and sleep architecture, emotional behavior (Banerjee et al., 2014; Amador et al., 2016; Mang et al., 2016), and mood regulation (Kishi et al., 2008; Chung et al., 2014).

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5.4.2 Role of REV-ERBα in peripheral tissues. The clock gene Rev-erbα also plays a significant role in various metabolic pathways. Reverbα is required for adipocyte differentiation (Chawla and Lazar, 1993; Fontaine et al., 2003; Wang and Lazar, 2008). Rev-erbα knock-out mice show increased adipose tissue mass and increased lipoprotein lipase (Lpl) expression in white adipose tissue, skeletal muscle and liver (Delezie et al., 2012). In vivo experiments in rat adipose tissues and in vitro experiments in 3T3-L1 cell lines show that rosiglitazone treatment activates PPARγ which further induces Rev-erbα expression through binding to the DR2 element in its promoter region (Fontaine et al., 2003; Laitinen et al., 2005). Brown adipose tissue (BAT) is a metabolically active tissue that participates in bodily thermogenesis. Thermogenesis is linked to the circadian clock mechanism via Rev-erbα. In 2013, Gerhart-Hines and colleagues demonstrated the role of Rev-erbα in BAT (Gerhart-Hines et al., 2013). REV-ERBα also promotes adipogenesis in BAT (Nam et al., 2015). Global Rev-erbα knock-out mice show increased tolerance to 4°C cold with increased Ucp1 expression in BAT (Delezie et al., 2012; Gerhart-Hines et al., 2013). In Bmal1 knock-out mice, 4°C cold exposure suppresses Rev-erbα expression in BAT (Li et al., 2013). In vivo studies in human subjects using 18-fluorodeoxyglucose (glucose analogue) show a diurnal rhythm of glucose uptake by BAT (Cypess et al., 2009; Virtanen et al., 2009). In mice, such a rhythm is abolished after deletion of Rev-erbα resulting in increased glucose uptake during daytime, but not at night (Gerhart-Hines et al., 2013). Skeletal muscles are metabolically active organs that participate in the homeostasis of glucose uptake and insulin sensitivity (Dyar et al., 2014). Rhythmic functioning in skeletal muscle is under the control of the skeletal molecular clock. Rev-erbα is expressed rhythmically in mouse skeletal muscle with a similar phase as in other peripheral clocks, such as liver and adipose tissues (Yang et al., 2006). REV-ERBα in the muscle represses a key gene, myoD which is essential for muscle cell differentiation as first shown in vitro in C2Cl2 cells (Downes et al., 1995). In addition, it represses the transcription of Bmal1 and Clock genes in muscle (Delezie et al., 2012). Rev-erbα knock-out mice displayed higher Lpl mRNA levels in skeletal muscle (Delezie et al., 2012), decreased oxidative function and decreased exercise, and increased autophagy (Woldt et al., 2013). Administration of the REV-ERBα agonist (SR9011) leads to an amplification in the circadian expression of genes involved in fatty acid oxidation and glycolysis in skeletal muscle (Solt et al., 2012). 39

The liver is the major organ for glucose and lipid metabolism and maintenance of whole body homeostasis. In the liver clock, REV-ERBα regulates expression of BMAL1 and CLOCK (Preitner et al., 2002; Delezie et al., 2012) and modulates body metabolism by regulating lipid, cholesterol and bile acid metabolism, liver gluconeogenesis, hepatic glycogen and circulating glucose, triglycerides and free fatty acids (Duez et al., 2008; Le Martelot et al., 2009; Delezie et al., 2012). REV-ERBα participates in regulation of the rhythmic expression of the rate limiting enzyme cholesterol-7α-hydroxylase (CYP7A1) required for cholesterol to bile acid metabolism. As discussed previously, REV-ERBα recruits the NCoR/HDAC3 complex for suppressing Bmal1 expression in the liver. REVERBα co-localizes with hepatic HDAC3 to regulate lipid metabolism and rhythmic histone acetylation. Accordingly, loss of Rev-erbα disturbs hepatic lipid homeostasis and causes hepatic steatosis (Feng et al., 2011; Bugge et al., 2012; Sun et al., 2012; Sun et al., 2013). REV-ERBα also may regulate the secretion of various endocrine hormones like insulin production by β cells and glucagon production by α cells in pancreatic islets, although conflicting results have been reported (Delezie et al., 2012; Vieira et al., 2012; Vieira et al., 2013).

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6. Aim of my thesis The aim of my thesis project was to focus on the relation between circadian clocks and clock genes on the one hand and feeding behaviour and diet composition on the other hand. Changes in feeding behaviour either due to an equal distribution of the daily meals over the 24-hour day/night cycle or a change in diet composition may result in disturbances of the circadian clock mechanism. Alternatively, mutations in clock genes may disturb feeding behaviour and, as a consequence, may alter energy metabolism. By investigating the central clock in the SCN and peripheral clocks in the liver, skeletal muscle, and brown adipose tissue under these different conditions we provided new insights into how the central and peripheral clocks of the body may affect whole body metabolism. Figure (14).

Figure 14: Schematic representation of the chapters representing the interactions between circadian clocks and feeding behaviour in a nocturnal rodent.

Chapter 2 Effects of ultradian feeding on central and peripheral clocks in mice In Chapter 2 we studied in mice the impact of an ultradian 6-meals-a-day feeding schedule on the regulation of the peripheral clock in the liver and the function of the central clock in the SCN, as well as on physiology and metabolism. As already discussed in the Introduction, restricted feeding has a major impact on the peripheral clocks, while caloric restriction also affects the SCN clock. Here in mice, we combined the two approaches, that is, ultradian restricted feeding associated or not with caloric restriction. The aim of this study was to differentiate the effects of timing and caloric restriction on the central and peripheral clocks of mice.

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Chapter 3 Effects of ultradian feeding on central and peripheral clocks in rats In Chapter 3 we continued the experiment described in Chapter 2 by performing a similar study in rats by exposing them to the 6-meals-a-day feeding schedule and focusing on the regulation of the central clock in the SCN and peripheral clocks in the liver, skeletal muscle, and brown adipose tissue. The aim of this study was to assess the effect of the 6meals-a-day feeding schedule in another species, the rat, and its impact on other peripheral tissues such as muscle and brown adipose tissue along with the liver in relation to circadian rhythmicity and metabolism.

Chapter 4 Differential effects of diet composition and timing of feeding behaviour on rat brown adipose tissue and skeletal muscle peripheral clocks In this chapter, we focused on the consequences of a high caloric free choice high-fat high-sugar (fcHFHS) diet along with time-restricted feeding on the daily expression of clock and metabolic genes in rats. The effects of restricted feeding along with a chow or hypercaloric diet have been studied extensively in the liver, but to a lesser extend in skeletal muscle (SM) and brown adipose tissue (BAT), in spite of their critical role in energy metabolism. The aim of this study was to understand the interactive effects of TRF and diet on whole body energy metabolism as well on the clock and metabolic gene expression in overlooked metabolically active tissues such as SM and BAT.

Chapter 5 Role of the clock gene Rev-erbα in feeding and energy metabolism The circadian control of feeding behaviour is still not fully understood. To better understand whether the circadian timing of feeding behaviour depends on clock genes, in Chapter 5 we focused on the characteristics of feeding behaviour in mice genetically ablated for the clock gene Rev-erbα. We performed a comparative study in mice with a global or brain specific deletion of Rev-erbα to better define the physiological role of Reverbα. More specifically, the aim of this study was to differentiate the central and peripheral effects of Rev-erbα in the control of feeding behaviour and energy metabolism.

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Chapter 2 Ultradian feeding in mice not only affects the peripheral clock in the liver, but also the master clock in the brain Satish Sen1,3,4, Hélène Raingard1, Stéphanie Dumont1, Andries Kalsbeek2,3,4, Patrick Vuillez1,4, Etienne Challet1,4

1

Regulation of Circadian Clocks team, Institute of Cellular and Integrative Neurosciences, UPR3212,

Centre National de la Recherche Scientifique (CNRS), University of Strasbourg, France. 2

Department of Endocrinology and Metabolism, Academic Medical Center (AMC), University of

Amsterdam, The Netherlands. 3

Hypothalamic Integration Mechanisms, Netherlands Institute for Neuroscience (NIN), Amsterdam, The

Netherlands. 4

International Associated Laboratory LIA1061 Understanding the Neural Basis of Diurnality, CNRS,

France and the Netherlands.

Corresponding author: Etienne Challet, INCI, CNRS UPR3212, 5 rue Blaise Pascal, 67084 Strasbourg, France. Tel: +33 388456693, e-mail: [email protected]

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Abstract Restricted feeding during the resting period causes pronounced shifts in a number of peripheral clocks, but not the central clock in the suprachiasmatic nucleus (SCN). By contrast, daily caloric restriction impacts also the light-entrained SCN clock, as indicated by shifted oscillations of clock (PER1) and clock-controlled (vasopressin) proteins. To determine if these SCN changes are due to the metabolic or timing cues of the restricted feeding, mice were challenged with an ultradian 6-meals schedule (1 food access every 4 h) to abolish the daily periodicity of feeding. Mice fed with ultradian feeding that lost 10% body mass (i.e., hypocaloric) became more diurnal, hypothermic in late night, and displayed larger (3.5-h) advance of body temperature rhythm, more reduced PER1 expression in the SCN, and further modified gene expression in the liver (e.g., larger phase-advance of Per2 and upregulated levels of Pgc-1α). While glucose rhythmicity was lost under ultradian feeding, the phase of daily rhythms in liver glycogen and plasma corticosterone (albeit increased in amplitude) remained unchanged. In conclusion, the additional impact of hypocaloric conditions on the SCN are mainly due to the metabolic and not the timing effects of restricted daytime feeding.

Keywords: Circadian rhythm, feeding, 6-meal schedule, clock gene, suprachiasmatic nucleus.

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INTRODUCTION Biological rhythms are under the control of circadian oscillators, including a master circadian clock located in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus and peripheral oscillators present in almost every cell of the body (Bray and Young, 2009). The underlying molecular mechanism of the clock is based on transcriptional and translational feedback loops consisting of positive and negative elements (Reppert and Weaver, 2001). When heterodimerized, the positive limb elements BMAL1 and CLOCK activate transcription of the negative elements (Period (Per)1, 2, 3, and Cryptochrome (Cry)1, 2) that in turn inhibit BMAL1/CLOCK transactivation. In parallel, other clock genes such as Rev-erbα, β and Ror α, β, whose transcription is also activated by BMAL1/CLOCK, modulate Bmal1 and Clock transcription (Preitner et al., 2002; Crumbley and Burris, 2011; Cho et al., 2012). Light perceived by the retina is the most potent synchronizer of the circadian rhythm produced by the molecular clock mechanism within the SCN. The molecular clockwork regulates the rhythmic transcription of clock-controlled genes, such as the gene coding for neuropeptide Arginine Vasopressin (Avp) (Jin et al., 1999). The output of the SCN controls the timing of peripheral clocks via nervous, hormonal and behavioral cues (Froy, 2011). Food access restricted to the usual resting period can phase-shift circadian oscillations in a number of peripheral organs and brain regions outside the SCN, while the SCN master clock remains synchronized to the light-dark cycle (Damiola et al., 2000; Stokkan et al., 2001; Feillet et al., 2008b). However, when daytime restricted feeding is combined with caloric restriction, the master clock is affected, as assessed by phase-advances in daily rhythms of body temperature, activity rhythm, and pineal melatonin, as well as by altered photic resetting (Challet, 2010). Moreover, daily caloric restriction leads to phase-shifts in daily oscillations of clock (PER1) and clock-controlled (AVP) proteins in the SCN (Mendoza et al., 2007b). To avoid the synchronizing effects of daily restricted feeding, a protocol has been developed using a feeding regimen of six 10-min food accesses equally distributed over 24 h (i.e., one 10-min meal every 4 h) (Kalsbeek and Strubbe, 1998). In nocturnal rats under light-dark conditions, this ultradian 6-meals-a-day feeding schedule does not modify the phase of locomotor activity rhythm, but if food access to the 6-meals is shortened to cause body mass loss, rats become partially active during daytime due to a phase-advance of the rest/activity rhythm (Mendoza et al., 2008b).

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One recent rat study showed that peripheral clock gene rhythms are still present during ultradian 6-meals-a-day feeding, despite changes in amplitude and phase (Su et al., 2016a). Another study suggested that in mice, the peripheral clocks remain unaffected by meal timing when each meal is given equally spaced either 2, 3, 4 or 6 times per day. However, if meal frequency is unevenly distributed, i.e., with unequal intervals between the meals, then the phase of peripheral clock genes changes, especially in the kidney. Moreover, that study also showed that ultradian 6-meal feeding coupled to caloric restriction was able to produce phase-advances of peripheral clocks inversely proportional to the degree of energy intake (Kuroda et al., 2012). In the present study, we aimed at investigating further whether it is the daily timing of feeding and fasting or metabolic cues associated with caloric restriction that affects the central and peripheral clocks. For that purpose, we challenged mice with a 6-meals-a-day feeding schedule (combined with isocaloric or hypocaloric conditions) and studied their behavioural and physiological changes, as well as expression of clock and clockcontrolled genes in the master clock and liver.

MATERIALS AND METHODS Animals and housing Seventy-six 5-week old male C57BL/6J mice (Janvier labs, Le Genest-Saint-Isle, France) were used for this study. The animals were housed in individual cages equipped with a wheel, at an ambient temperature of 23 ± 2°C under 12:12 h light-dark conditions (lights on at 7:00 AM (defining Zeitgeber Time (ZT) 0) and off at 19:00 PM (=ZT12)). In a group of 46 animals, access to food was automatically controlled by electronic timers for six cages at a time. Thirty animals served as controls and had ad libitum access to food. 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 in advance by the Regional Ethical Committee of Strasbourg for Animal Experimentation (AL/50/57/02/13) and in compliance with the ethical standards of the journal (Portaluppi et al., 2010).

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Surgery Mice were anesthetized with isoflurane (Vetflurane, Virbac 3% powered by 0.2 l/ min O2) to implant a transponder (Minimitter, Vitalview, Sunriver, OR, USA) in the abdominal cavity to record body temperature and general cage activity. The abdomen was shaved and sprayed with antiseptic (DermaSpray, Bayer) before an incision in the skin and muscle (810 mm) was made. Once the transponder was inserted into the abdominal cavity, the muscle layer was stitched with surgical sutures (Filapeau, 3.0) and anti-inflammatory medication was provided in drinking water (Metacam, 0.2 mg/ml, 0.1ml) for 2 days. Experimental procedure After surgery, the animals were placed in experimental cages for 2 weeks with drinking water and food ad libitum. After this, mice were habituated to an ultradian schedule of six meals each day with one food access every 4 h (ZT2, ZT6, ZT10, ZT14, ZT18 and ZT22). Access to food during restricted feeding was set automatically by the Food Planning system based on a food basket allowing and preventing food access in the low and upper position, respectively (Intellibio, Seichamps, France). Lifting and fall of the food basket being associated with a brief motor noise, these auditory cues may have signaled food availability to the mice. The smaller mesh size of the trough compared to the size of the food pellets prevented any food hoarding in the cage. Duration of food access was reduced every 4 days gradually from 6 x 1 h, via 6 x 30 min, 6 x 20 min to 6 x 15 min. This protocol was based on previous studies in rats (Kalsbeek and Strubbe, 1998; Mendoza et al., 2008b). The fact that mice were fed every day at the same times has probably improved their ability to adjust to ultradian feeding, as opposed to irregular meal times (Valle, 1981). Food intake during daytime and nighttime was measured twice (at the steps of 6 x 1 h and 6 x 15 min) to evaluate the day-night pattern of food intake. Body mass was measured weekly. At the end of two weeks of feeding according to the 6 x 15 min protocol, two groups were categorized according to individual adaptation to the paradigm, eventually leading to body mass loss. A cut-off at 10% body mass loss allowed to distinguish an isocaloric group including animals with less than 10% of body mass loss (mean: 5.4 ± 0.5%; n total=24; n=4 per ZT) and a hypocaloric group in which animals lost 10% or more (up to 25%) body mass (mean: 15.5 ± 1.1%; n total=22; n=3-4 per ZT). Animals of the control group were kept with food and water ad libitum (n=5 per ZT). 67

Immunohistochemistry At the end of the experiment, animals were sacrificed with an overdose of pentobarbital. Mice fed with ultradian 6-meals schedule were sampled every 4 h between food accesses (i.e., ZT0, ZT4, ZT8, ZT12, ZT16 and ZT20) to limit direct effects of feeding while avoiding prolonged fasting. Control mice fed ad libitum were sacrificed at the same times. Blood was sampled by intracardiac puncture, liver was sampled in the right lobe, and the heart was perfused with 50 mL of 0.9% saline followed by 50 mL of 4% paraformaldehyde in phosphate buffer (0.1 M, pH 7.4). Brains were removed, postfixed overnight in 4% paraformaldehyde (4°C) and transferred to a cryoprotectant buffered sucrose solution (30% at 4°C) for at least 24 h till brains sank to the bottom due to the sucrose density gradient. Brains were then frozen in isopentane around -50°C and stored at -80°C. Five series of 30-μm coronal SCN sections were prepared on a cryostat and collected in Phosphate-Buffered Saline (0.1 M PBS, 1x) and washed with 1x Tris Buffer Saline pH 7.6 (0.1 M TBS 1x). Then sections were incubated in 3% H2O2 in TBS (30 min) to suppress endogenous peroxidase activity, thereby reducing background staining. Again brain sections were rinsed in TBS 1x. Brain sections were then transferred in a solution containing 10% normal serum (either goat or horse according to the host species of the primary antibody) and Triton X-100 (0.1 %) in TBS for 2 h, followed by incubation in the primary antibody (48 h at 4°C). We used rabbit polyclonal anti arginine-vasopressin (AVP) (1:20000, Truus, a gift from Dr. Ruud Buijs, Netherlands Institute for Brain Research, Amsterdam, the Netherlands), goat polyclonal anti-PER1 (1:750; SC-7724, Santa Cruz Biotechnologies, Santa Cruz, CA, USA) and rabbit polyclonal anti-PER2 (1:3000, #PER-21A; Alpha Diagnostic International, San Antonio TX, USA; note that for anti-PER2 immunohistochemistry, PBS indicated below was always replaced with TBS). The sections were washed in PBS 1x, then incubated (2 h at 4°C) with biotinylated goat anti-rabbit IgG (1:500, PK6101; Vectastain Standard Elite ABC Kit Vector Laboratories, Inc., Burlingame, CA, USA) for AVP and PER2 and with biotinylated anti-goat IgG made in horse (1:500, BA-9500; Vector labs) for PER1 immunostaining. After this, sections were rinsed in PBS 1x and incubated (2 h) in a solution containing avidin–biotin peroxidase complex (Vectastain Elite ABC kit; Vector Laboratories Inc.). Following incubation with ABC reagents, sections were rinsed 4 times in PBS, and incubated with H2O2 (0.015%, Sigma-Aldrich, St Louis, MO, USA) and 3,3’diaminobenzidine 68

tetrahydrochloride (0.5 mg/ml, Sigma-Aldrich) diluted in water. Thereafter, sections were rinsed with PBS, wet mounted on slides coated with gelatin, dehydrated through a series of alcohols, soaked in xylene, and cover slipped. Photomicrographs were taken on Leica DMRB microscope (Leica Microsystems) with an Olympus DP50 digital camera (Olympus France). The number of immunopositive cells was counted on one section in both SCN’s and averaged. mRNA extraction and quantitative real-time PCR RNA was extracted from frozen liver samples by homogenizing liver samples in lysis buffer supplemented with β-mercaptoethanol and using absolutely RNA miniprep kit (Agilent Technologies, USA. The samples were purified by precipitation with sodium acetate and isopropyl alcohol. The quality of RNA was measured on NanoDrop ND-100 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA; A260/A280, and A260/A230 values were > 1.8) RNA integrity was assessed using (Agilent RNA 6000 Nano Kit) on Aligent 2100 bio-analyzer for all the liver samples (RIN Value were >7) bio-analyzer. cDNA was synthesized with the High Capacity RNA to cDNA kit (Applied Biosystem, Foster city CA, USA) using 1µg of RNA. Measurement of relative abundance was performed by real-time PCR analysis using 1X of TaqMan Gene Expression Master Mix (Life Technologies, Foster city, CA, USA). The following TaqMan probes (Per2: Mm00478113_m1,

Clock:

Mm00455950_m1,

Sirt1:

Mm00490758_m1,

Fgf21:

Mm00840165_g1, Nr1d1 (Rev-erb α): Mm00520708_m1, Pparα: Mm01208835 m1 and Pgc-1α: Mm00440939_m1) were used for all the genes with 1µl of cDNA in the reaction mixture of 20 μl. Each reaction PCR was done in duplicate. A dilution curve was prepared of pooled cDNA samples using log10 standards to calculate the amplification efficiency for each primer set (values were between 1.85-1.99). Data were normalized to Tbp (Mm00446971_m1) and analysed the comparative cycle threshold (Ct) method RQ= 2∆∆Ct. ∆∆Ct =∆Ct sample- ∆Ct reference (Pfaffl, 2001) with efficiency corrections. Transcript levels were calculated relative to the mean of ZT 0 samples. Plasma metabolic parameters Plasma samples were obtained after centrifugation of fresh blood collected with 4% EDTA

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(10 µL for 1 mL of blood) and centrifuged for 10 min (5000 rpm at 4°C). Plasma glucose was evaluated with GOD-PAP kit (Biolabo, Maizy, France). The ACS-ACOD method (NEFA-HR2; Wako, Osaka, Japan) was used for assaying plasma non-esterified fatty acids (NEFA). Plasma concentrations of corticosterone were determined by a Rat/Mouse Corticosterone EIA kit (AC-14F1, IDS EURL, Paris, FRANCE). The limit of sensitivity of the assay was 0.55 ng/mL. Hepatic glycogen assay Samples of fresh liver were flash-frozen in liquid nitrogen. Hepatic glycogen was quantified according to the method developed by Murat and Serfaty (Murat and Serfaty, 1974). Statistical analysis Data are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed by SigmaPlot (version 12, SPSS Inc, Chicago, IL, USA). Significance was defined at p