Julien Delezie

Stéphanie Dumont

Cristina Sandu

Sophie Reibel

Paul Pevet

Etienne Challet
email: challet@inci-cnrs.unistra.fr

Rev-erbα in the brain is essential for circadian food entrainment

Foraging is costly in terms of time and energy. An endogenous food-entrainable system allows anticipation of predictable changes of food resources in nature. Yet the molecular mechanism that controls food anticipation in mammals remains elusive. Here we report that deletion of the clock component Rev-erbα impairs food entrainment in mice. Rev-erbα global knockout (GKO) mice subjected to restricted feeding showed reduced elevations of locomotor activity and body temperature prior to mealtime, regardless of the lighting conditions. The failure to properly anticipate food arrival was accompanied by a lack of phase-adjustment to mealtime of the clock protein PERIOD2 in the cerebellum, and by diminished expression of phosphorylated ERK 1/2 (p-ERK) during mealtime in the mediobasal hypothalamus and cerebellum. Furthermore, brain-specific knockout (BKO) mice for Reverbα display a defective suprachiasmatic clock, as evidenced by blunted daily activity under a light-dark cycle, altered free-running rhythm in constant darkness and impaired clock gene expression. Notably, brain deletion of Rev-erbα totally prevented food-anticipatory behaviour and thermogenesis. In response to restricted feeding, brain deletion of Rev-erbα impaired changes in clock gene expression in the hippocampus and cerebellum, but not in the liver. Our findings indicate that Rev-erbα is required for neural network-based prediction of food availability.

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 . Here, we evaluated the role of Rev-erbα, a gear of the circadian clockwork and a regulator of glucose and lipid metabolism 4,24-30 , in the ability of mice to predict feeding time. We hypothesized that brain expression of REV-ERBα may be required to link circadian feeding cues to the central regulation of behaviour and physiology. We show that both global and brain-specific deletions of Rev-erbα profoundly altered the expression of food-anticipatory components. This study gives evidence that Rev-erbα in the central nervous system is essential to shape the 24-hour pattern of activity in conditions of limited food access.

Results

Food anticipation is reduced in global Rev-erbα KO mice in light-dark conditions. No significant differences were found between genotypes fed ad libitum regarding body mass and food intake, 24-h body temperature rhythm and daily general locomotor activity (Fig. 1B, as previously shown in ref. 30). We however observed that global deletion of Rev-erbα tended to reduce spontaneous wheel-running activity (10399 ± 2117 vs. 7174 ± 3184 revolutions/24 h in WT and GKO mice, respectively; t (13) = 0.9, p = 0.4) as already found in [START_REF] Woldt | Rev-erb-alpha modulates skeletal muscle oxidative capacity by regulating mitochondrial biogenesis and autophagy[END_REF] . In particular, heterogeneity in wheel-running behaviour ranged from robust rhythms to fragmented profiles, we thus excluded this parameter in most of the results presented thereafter.

To test whether Rev-erbα deletion changes the ability to anticipate mealtime, global knockout (GKO) and wild-type (WT) mice were subjected to 6-h restricted feeding. As expected, WT mice expressed a strong FAA 1 h before meal access, illustrated by significant increase of general activity at that time compared to the other time-points (Effect of ZT: F (3,59) = 20.1, p < 0.001; [Genotype x ZT] interaction: F (3,59) = 5.2, p < 0.01 Fig. 1A-C). In contrast, GKO mice did not show a significant increase of general activity in anticipation of mealtime. The amount of general activity in GKO mice 1 h before meal access was reduced compared to WT mice (Tukey HSD post-hoc test, p < 0.05; Fig. 1A-C). Moreover, the sharp preprandial rise in body temperature seen in WT animals was not observed in GKO mice ([Genotype x ZT] interaction: F (3,59) = 2.8, p < 0.05; Fig. 1B,C). Of note, both genotypes showed a similar body mass loss after one week of restricted feeding (WT = -8.1 ± 2.1 vs. GKO = -9.5 ± 3.1%; t (13) = 0.3, p = 0.7.). Lastly, restricted feeding induced a 6-h phase shift of clock-related mRNA expression in the liver of WT animals, in line with previous studies [START_REF] Damiola | Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus[END_REF][START_REF] Tahara | Effects of medial hypothalamic lesions on feeding-induced entrainment of locomotor activity and liver Per2 expression in Per2::luc mice[END_REF][START_REF] Feillet | Lack of food anticipation in Per2 mutant mice[END_REF] . Food-induced phase-adjustment of liver oscillations however also occurred in GKO mice in spite of up-regulated Clock and Bmal1 expression (Fig. 2A-C). Conversely, global deletion of Rev-erbα abolished the anticipatory peak of corticosterone ([Genotype x ZT] interaction: F (3,38) = 4.1 p = 0.01; Fig. 2D), considered as a food-anticipatory component [START_REF] Nelson | Circadian rhythms in mice fed a single daily meal at different stages of lighting regimen[END_REF][START_REF] Honma | Feeding-associated corticosterone peak in rats under various feeding cycles[END_REF] . Together, these findings indicate that behavioural and physiological components of meal anticipation are clearly blunted in GKO mice under a light-dark cycle.

FAA in global Rev-erbα KO mice is altered in constant darkness. To avoid a potential masking effect of light known to attenuate activity in nocturnal animals [START_REF] Patton | Photic and pineal modulation of food anticipatory circadian activity rhythms in rodents[END_REF] , which can account for the reduced food anticipation in Rev-erbα-deficient mice, we exposed WT and GKO mice to constant dark (DD) conditions or to skeleton photoperiod. In DD conditions with animals fed ad libitum, the global deletion of Rev-erbα shortened the free-running period (WT = 24.1 ± 0.1 h vs. GKO = 23.2 ± 0.1 h; t (9) = 13.9, p < 0.001; as in ref. 4). Furthermore, mealtime was shifted (i.e., 6-h phase-delayed) to investigate how FAA reappears at a new feeding time. In both DD conditions (with or without shift of mealtime) and skeleton photoperiod, we did not detect more FAA in GKO mice than in light-dark (LD) conditions (Figs 3A,B and 4A,B).

Hence, regardless of the lighting conditions, the time of the day when food was provided, global deletion of Rev-erbα markedly reduces FAA.

Feeding time differently affects PER2 and p-ERK expressions in the cerebellum of global

Rev-erbα KO mice.

A number of extra-SCN oscillators sensitive to feeding cues could be part of the food-entrainable network [START_REF] Feillet | Forebrain oscillators ticking with different clock hands[END_REF][START_REF] Mendoza | The cerebellum harbors a circadian oscillator involved in food anticipation[END_REF][START_REF] Angeles-Castellanos | c-Fos expression in hypothalamic nuclei of food-entrained rats[END_REF] . We therefore investigated PER2 expression and used the phosphorylated form of the Extracellular signal Regulated Kinases I/II (p-ERK) as a marker of neuronal activation in response to restricted feeding in hypothalamic nuclei, including dorsomedial (DMH), arcuate (ARC), suprachiasmatic (SCN), ventromedial (VMH) and paraventricular (PVN) nuclei, and the cerebellum. When food was provided ad libitum, PER2 expression was rhythmic in all and most evaluated structures in WT and GKO mice, respectively (Supplemental Table S1). Similar timing was found in the SCN in both genotypes. There was a trend for phase-advanced peak in the ARC and PVN of GKO mice (and possibly also in VMH and cerebellum) compared to controls (Fig. 5A-C; Supplemental Table S1). Expression of p-ERK was rhythmic in the cerebellum with close timing in GKO and WT mice fed ad libitum (Supplemental Table S2). In the ARC, p-ERK expression was rhythmic in both genotypes, while in other hypothalamic regions (VMH, DMH, PVN and SCN), no significant cosinor regression was detected (Fig. 5D,E; Supplemental Table S2). Furthermore, except for the cerebellum, the daily pattern of p-ERK expression did not exactly match between WT and GKO, with apparent phase-changes and/or dampened amplitude (Fig. 5F; Supplemental Table S2).

Besides the SCN insensitive to mealtime, adaptation of PER2 oscillations to restricted feeding was observed in most hypothalamic nuclei (ARC, VMH and PVN) of WT mice, as shown by an earlier PER2 expression that increased during food access (Fig. 5A,B, Supplemental Table S1). Close phase-advances were found for ARC and PVN (and a trend for VMH) of GKO mice. By contrast, PER2 expression in the cerebellum was markedly phase-advanced in food-restricted WT mice, but not in GKO mice (Fig. 5C, Supplemental Table S1). In response to daytime feeding, p-ERK expression was phase-advanced in the ARC of WT mice, while this change was hardly detectable in GKO mice (Fig. 5D,E, Supplemental Table S2). In the cerebellum, p-ERK expression in WT animals was clearly up-regulated before and during food access, while in GKO mice, the daily trough of p-ERK expression occurred prior to mealtime (Fig. 5F).

In sum, neuronal activation during food anticipation is less marked in the hypothalamus and cerebellum of GKO mice, which may contribute to their reduced FAA. Furthermore, phase-adjustment of the circadian clockwork to feeding time does not require the expression of Rev-erbα in hypothalamic and hepatic oscillators, whereas it seems indispensable for cerebellar clock adjustment.

Circadian phenotype of brain specific KO for Rev-erbα fed ad libitum.

To further evaluate the central contribution of Rev-erbα to food entrainment, we generated brain-specific KO mice. Moreover, the use of conditional KO mice may circumvent potential confounding effects of global clock gene deletion on whole-body energy homeostasis and therefore on food entrainment (e.g., refs 37-39). The NesCre-induced recombination of the floxed Rev-erbα allele was confirmed from multiple brain regions of NesCre-Rev-erbαΔ/-mice (noted BKO below) by qPCR analysis, in comparison to NesCre-Rev-erbΔ /+ mice (noted CTRL thereafter). We observed a nearly non-existent expression of the Rev-erbα transcript in the cerebellum (Effect of Genotype, F (1,21) = 16.1,

Figure 2. Alterations in hepatic expression of clock genes and plasma corticosterone in food-restricted

Rev-erbα GKO mice. Expression of Bmal1 (A), Clock (B), Per2 (C) under conditions of food ad libitum (left panel) and restricted feeding (right panel). (D) Plasma corticosterone levels in both food ad libitum (left) and restricted feeding conditions (right). Daytime and nighttime are indicated by white and black rectangles, respectively, on the X axis. Food access during restricted feeding is depicted by a grey rectangle. Data for ZT0 are double-plotted. *p < 0.05 for GKO vs. WT animals at a given time-point as determined by 2-way ANOVA and Tukey HSD post-hoc test; a p < 0.05 for restricted feeding vs. food ad libitum at a given time-point in wild-type (WT) mice (2-way ANOVA and Tukey post-hoc test); b p < 0.05 for restricted feeding vs. food ad libitum at a given time-point in Rev-erbα global knock-out (GKO) mice (2-way ANOVA and Tukey post-hoc test). a.u., arbitrary units. p < 0.001). In addition, we performed exploratory analyses in other brain regions. Very efficient NesCre-driven recombination was found in the hippocampus (Effect of Genotype, F (1,18) = 286.9, p < 0.001; Fig. 6A), nucleus accumbens, striatum, and motor cortex (data not shown). Down-regulation of Rev-erbα expression was less strong in the SCN and ARC of BKO mice, although Rev-erbα mRNA levels were still very significantly reduced and did not cycle between ZT2 and ZT10 (Effect of Genotype: F (1,18) = 24.0 and F (1,18) = 102.9, respectively in the SCN and ARC, p < 0.001 for both structures; Fig. 6A). Of note, one mouse heterozygous for NesCre and carrying both a KO and a floxed allele as confirmed by pre-weaning and post-death genotyping was excluded from the analysis, since Rev-erbα mRNA levels were normally expressed in the SCN of this mouse, which furthermore demonstrated a normal circadian phenotype (data not shown). These data indicate that the NesCre-driven recombination is not fully consistent amongst brain regions, in line with previous studies that used the same strategy [START_REF] Mieda | Bmal1 in the nervous system is essential for normal adaptation of circadian locomotor activity and food intake to periodic feeding[END_REF] . As a result of Rev-erbα knockdown in the brain, we detected a significant elevation of the Bmal1 transcript at both time points, regardless of the brain regions considered (Effect of Genotype, p < 0.05 in the cerebellum, SCN, ARC and hippocampus. For details of Tukey HSD post-hoc tests, see Fig. 6B). Conversely, Per2 mRNA levels were less affected in most brain nuclei (Effect of Genotype: p > 0.05 in the cerebellum, SCN and hippocampus; and A few days in ad libitum (LD) feeding conditions are illustrated before transfer to DD. During restricted feeding, food access is indicated by the grey rectangle. After a first period of food restriction at a specific circadian phase, mealtime was shifted (i.e., 6-h phase-delayed). (B) Double-plotted actograms of wheel-running activity from two WT mice (upper panel) and two GKO mice (lower panel) exposed to skeleton photoperiod. Note that the 12-h light and dark phases are represented respectively by white and black bars above the actograms, and that the two 1-h light pulses are indicated by yellow lines. A few days of ad libitum (AL) feeding conditions is illustrated before food restriction (RF, grey rectangle). significant in the ARC: F (1,18) = 18.9, p < 0.01 Fig. 6C), while we observed reduced amplitude of Per2 between both time points in several brain regions of BKO mice, including the cerebellum (Effect of ZT: F (1,18) = 38.1, p < 0.01; [Genotype x ZT] interaction: F (1,18) = 7.6, p = 0.01; Fig. 6C).

In LD conditions with food ad libitum, brain deletion of Rev-erbα did not affect body mass (21.6 ± 0.9 vs. 23.7 ± 1.2 g in BKO and CTRL mice, respectively; t (11) = 1.4, p = 0.18). All control groups (NesCre-Rev-erbΔ /+, Rev-erbαflox/+ , Rev-erbαflox/-) showed similar circadian organization and levels of both daily general locomotor and wheel-running activities (data not shown). We however observed a virtual absence of wheel use in BKO mice, thus precluding reliable comparison of wheel-running activity with the other genotypes (data not shown). The amplitude of day-night changes in general locomotor activity was drastically reduced without brain expression of Rev-erbα. In some animals, activity was rather fragmented and bursts of activity occurred mainly at the day-night/night-day transitions (Fig. 7A,B). The circadian general locomotor activity in BKO mice was further altered in DD conditions with a strong heterogeneity: three BKO mice were behaviourally arrhythmic, one exhibited a shorter and the other two had a longer free-running period compared to CTRL mice (Fig. 7A,B). These changes highlight major changes in the light-entrainable suprachiasmatic clock.

Diminished Rev-erbα expression in the brain accounts for altered food entrainment. To determine whether Rev-erbα in the brain is required for circadian food entrainment, we challenged BKO mice with 6-h restricted feeding in LD conditions. All food-restricted control mice rapidly developed FAA 1 to 2 h prior to mealtime (Fig. 8A,B), the peak of FAA 1 h before food access representing about 10 to 12% of daily total activity in all control groups. By contrast, BKO mice did not readily express behavioural activation 1 h before food arrival, FAA being hardly detectable above their mean level of daily activity ([Genotype x ZT] interaction: F (9,83) = 6.9, p < 0.001; For details of Tukey HSD post-hoc tests, see Fig. 8B). In accordance with behavioural data, the peak of thermogenesis in anticipation was totally abolished in BKO mice (Effect of genotype: F (3,83) = 6.2, p < 0.01; [Genotype x ZT] interaction: F (9,83) = 7.9, p < 0.001; Fig. 8B). Moreover, like GKO mice, food-restricted BKO mice were able to maintain body temperature levels close to those measured in ad libitum conditions. During the 6-h food access, the daily amount of food eaten was smaller in BKO (NesCre-Rev-erbΔ /-) mice compared to CTRL (NesCre-Rev-erbΔ /+ ) animals (2.9 ± 0.2 g vs 3.8 ± 0.1 g, respectively; t (11) = 3.9, p < 0.01), while body mass during and at the end of the 2 wks of restricted feeding was not significantly different between the two genotypes (19.4 ± 1.0 vs. 22.4 ± 1.0 g, respectively; t (11) = 2.1, p = 0.06) as was body mass loss (-10.0 ± 2.3% vs. -5.3 ± 0.9; respectively; t (11) = 2.0, p = 0.07).

Interestingly, in food-restricted CTRL mice, Rev-erbα expression was up-regulated at ZT2 in the ARC, Hipp and CRB, while Rev-erbα levels were as low as during ad libitum conditions in brain areas of BKO mice (for details, see Fig. 6A). Expression of Bmal1 and Per2 was not affected in the SCN of food-restricted BKO and CTRL mice in comparison with conditions of food ad libitum (Fig. 6B,C) and Bmal1 was still up-regulated in the SCN of food-restricted BKO animals (Effect of Genotype: F (1,13) = 43.1, p < 0.001). Besides Rev-erbα, morning and evening expression of Per2 and/or Bmal1 was also altered in the ARC, hippocampus and CRB of CTRL mice in response to restricted feeding (Fig. 6). In the ARC, levels of Per2 and Bmal1 were up-regulated in food-restricted BKO mice as compared to CTRL animals (Effect of Genotype: F (1,15) > 12, p < 0.01 for both genes), while phase-changes were close between genotypes ([Genotype x ZT] interaction: F (1,15) < 0.1, p > 0.8) for both genes). In the hippocampus, Bmal1 mRNA levels were down-regulated in food-restricted mice (Fig. 6), albeit at higher levels in BKO as compared to CTRL mice (Effect of Genotype: F (1,16) = 11.1, p < 0.01). Per2 expression in the hippocampus was differentially affected by mealtime according to the genotype ([Genotype x ZT] interaction: F (1,16) = 7.7, p = 0.01): Per2 mRNA levels were increased in the morning only in BKO and in the evening only in CTRL mice. Finally, in addition to increased Rev-erbα levels in response to mealtime in the cerebellum of CTRL mice, Per2 expression was also up-regulated, indicating phase-sensitivity to mealtime. In the cerebellum of BKO mice, the morning levels of Per2 expression was similar between restricted feeding and ad libitum food, suggesting impaired phase-adjustment of the cerebellar clock to mealtime (Fig. 6C). In contrast, except for Per2 expression in BKO animals that was more diminished in the morning, changes in clock gene expression in the liver of food-restricted mice were close between BKO and CTRL groups (Supplemental Fig. 1), indicating that the phase-adjustment of the liver clock to mealtime was essentially unaltered in BKO mice.

We also tested the reappearance of FAA: food-restricted mice were transferred back to conditions with ad libitum food for 5 days and then challenged with a 24-h fast. Among the fasted control groups, Rev-erbα flox/+ mice (i.e., mice that possess both a WT allele and a non-recombined floxed allele) showed a strong increase in locomotor activity before expected food access while the progressive elevation of FAA was much slighter in the other controls (Effect of genotype: F (3,83) = 6.3, p < 0.01; [Genotype x ZT] interaction: F (9,83) = 2.7, p = 0.01; Fig. 8C). On the other hand, a rise in body temperature before expected mealtime was observed in all control groups (Effect of genotype: F (3,83) = 6.1, p < 0.01; [Genotype x ZT] interaction: F (9,83) = 2.9, p < 0.01; Fig. 8C). Notably, neither FAA nor food-anticipatory thermogenesis could be observed in fasted BKO mice (Fig. 8C). Overall, the lack of behavioural and physiological components of food anticipation in a brain-specific KO model reveals that central Rev-erbα expression is crucial for proper food entrainment.

Discussion

The role of clock genes in the food-entrainable network has been assessed by challenging clock-compromised mice with time-restricted feeding. Some studies suggest that circadian food anticipation relies on a clock gene-dependent mechanism that at least involves the Period1-3, Bmal1, Cry1-2 and Npas2 genes [START_REF] Takasu | Circadian regulation of food-anticipatory activity in molecular clock-deficient mice[END_REF][START_REF] Feillet | Lack of food anticipation in Per2 mutant mice[END_REF][START_REF] Mieda | Bmal1 in the nervous system is essential for normal adaptation of circadian locomotor activity and food intake to periodic feeding[END_REF][START_REF] Chavan | Liver-derived ketone bodies are necessary for food anticipation[END_REF][START_REF] Mendoza | Behavioural food anticipation in clock genes deficient mice: confirming old phenotypes, describing new phenotypes[END_REF][START_REF] Dudley | Altered patterns of sleep and behavioral adaptability in NPAS2-deficient mice[END_REF][START_REF] Pendergast | Period determination in the food-entrainable and methamphetaminesensitive circadian oscillator(s)[END_REF][START_REF] Li | Sex-related difference in food-anticipatory activity of mice[END_REF] . Others indicate that contribution of clock genes to food entrainment is mild [START_REF] Horikawa | Rapid damping of food-entrained circadian rhythm of clock gene expression in clock-defective peripheral tissues under fasting conditions[END_REF][START_REF] Iijima | Altered food-anticipatory activity rhythm in Cryptochrome-deficient mice[END_REF] , or even non-significant [START_REF] Storch | Daily rhythms of food-anticipatory behavioral activity do not require the known circadian clock[END_REF][START_REF] Pendergast | Robust food anticipatory activity in BMAL1-deficient mice[END_REF][START_REF] Pitts | Food-entrained circadian rhythms are sustained in arrhythmic Clk/Clk mutant mice[END_REF] . Here we give evidence that central Rev-erbα expression is necessary for an animal to predict the time of food availability. In conditions of limited food access, global Rev-erbα deletion induces a significant reduction of FAA in LD conditions. This dampened FAA was further confirmed in DD conditions or skeleton photoperiod. In accordance with behavioural results, the food-anticipatory thermogenesis was nearly absent in GKO mice. By contrast, the postprandial peak of body temperature matched between WT and GKO mice, indicating similar food processing. In addition, we observed similar body mass values and no significant reduction or delay in food intake between food-restricted WT and GKO animals (data not shown), thus excluding poor health status as a cause of reduced food anticipation. Lastly, corticosterone levels were not elevated in anticipation of daytime feeding in GKO mice. Hence, the overall altered food-entrained physiology of GKO mice supports an essential role for Rev-erbα in food entrainment.

The liver clock is sensitive to feeding cues [START_REF] Feillet | Lack of food anticipation in Per2 mutant mice[END_REF] and Rev-erbα may play a role in that regard [START_REF] Tahara | Refeeding after fasting elicits insulin-dependent regulation of Per2 and Reverbalpha with shifts in the liver clock[END_REF] . Phase-adjustment of hepatic clock actors to feeding time was however preserved in GKO mice. This indicates that disrupted FAA is not the consequence of impaired liver clock entrainment to scheduled feeding. Of note, the expression of clock-related genes in the liver of Per2 mutant mice is also shifted in response to daytime feeding [START_REF] Feillet | Lack of food anticipation in Per2 mutant mice[END_REF] . It is thus likely that the liver clock is robust to clock perturbations on the long term and may rely on metabolic cues to adjust the phase of circadian mRNA expression in conditions of time-restricted food access.

PER2 expression has been shown to respond to feeding time in hypothalamic oscillators [START_REF] Feillet | Forebrain oscillators ticking with different clock hands[END_REF] . Our results show that PER2 expression in the hypothalamic nuclei studied was not significantly different between both genotypes, either fed ad libitum or challenged with restricted feeding. Therefore, the diminished capability of food entrainment in GKO mice is not correlated with altered PER2 cycling in hypothalamic areas. Among hypothalamic oscillators, several studies have been focused on the DMH, thought to be part of the food-entrainable network [START_REF] Gooley | The dorsomedial hypothalamic nucleus is critical for the expression of food-entrainable circadian rhythms[END_REF][START_REF] Acosta-Galvan | Interaction between hypothalamic dorsomedial nucleus and the suprachiasmatic nucleus determines intensity of food anticipatory behavior[END_REF][START_REF] Verhagen | Anticipation of meals during restricted feeding increases activity in the hypothalamus in rats[END_REF] , but see also [START_REF] Landry | Evidence for time-of-day dependent effect of neurotoxic dorsomedial hypothalamic lesions on food anticipatory circadian rhythms in rats[END_REF][START_REF] Moriya | The dorsomedial hypothalamic nucleus is not necessary for food-anticipatory circadian rhythms of behavior, temperature or clock gene expression in mice[END_REF] ). Here we did not detect sizeable PER2 immunostaining in the DMH, as previously reported [START_REF] Feillet | Forebrain oscillators ticking with different clock hands[END_REF] . Together, these data indicate that Rev-erbα may be dispensable for adjustment of hypothalamic clock gene oscillations as in the liver, although these results do not rule out a role for hypothalamic nuclei in food entrainment.

Increased expression of c-FOS has been observed before and/or after mealtime in several hypothalamic nuclei, including the DMH and the VMH [START_REF] Gooley | The dorsomedial hypothalamic nucleus is critical for the expression of food-entrainable circadian rhythms[END_REF][START_REF] Angeles-Castellanos | c-Fos expression in hypothalamic nuclei of food-entrained rats[END_REF][START_REF] Ribeiro | Two forces for arousal: Pitting hunger versus circadian influences and identifying neurons responsible for changes in behavioral arousal[END_REF] . By studying the activation marker p-ERK, we showed for the first time that its daily pattern of expression was adjusted to feeding time in all hypothalamic nuclei of WT animals. On animals and NesCre-Rev-erbαΔ/-(BKO) mice under LD conditions with food provided ad libitum and then restricted to 6 h per day (grey box, food was provided from ZT6 to ZT12). Note that on the last day of restricted feeding (day 24), food was given 6 h later (i.e., ZT12) than before. Thereafter, food was provided ad libitum except from day 28 (ZT12) to day 29 (ZT12) during which no food was given ("fasting test", see arrow on Y axis). (B,C, upper panels) Normalized activity profiles and body temperature raw data representing the average of 10 days during restricted feeding conditions (B) and fasting test (C) in NesCre-Rev-erbαΔ/+ (CTRL, blue circle), NesCre-Rev-erbαΔ/-(BKO, red circle), Rev-erbαflox/+ (Flox/+ , orange down-pointing triangle) and Rev-erbαflox/-(Flox/-, green up-pointing triangle) mice. Daytime and nighttime are indicated by white and black bars above the graphs, respectively. The period of food access during RF is represented by the grey rectangle. (B,C, lower panels). Statistical analysis (2-way ANOVA) was performed on percent of FAA over total daily activity and body temperature data from 4 h to 1 h before expected mealtime. a p < 0.05 for BKO vs. other groups, b p < 0.05 for BKO vs. Flox/+ and Flox/1, c p < 0.05 for CTRL vs. other groups, d p < 0.05 for Flox/+ vs. other groups, e p < 0.05 for BKO vs. Flox/+ , and f p < 0.05 for BKO vs. Flox/-. the contrary, p-ERK expression was affected by global Rev-erbα deletion in both feeding conditions and no peak of p-ERK expression was detected in GKO in anticipation of mealtime. This indicates that feeding/homeostatic signals are not similarly integrated in hypothalamic areas of GKO mice compared to WT mice, while PER2 phase-adjustment to scheduled feeding remains unaffected in the absence of Rev-erbα and the consecutive alteration of p-ERK activation. Distinct cellular pathways, therefore, may be involved in the entrainment of brain oscillators and in the induction of immediate early genes in response to feeding-related cues.

On the other hand, additional brain areas may be essential to drive FAA. In support to that view, brain-specific KO of Bmal1 37 , but not forebrain-specific deletion [START_REF] Izumo | Differential effects of light and feeding on circadian organization of peripheral clocks in a forebrain Bmal1 mutant[END_REF] , reduces FAA, suggesting that midbrain or hindbrain structures may modulate FAA. The cerebellum has recently been shown to harbor a circadian oscillator sensitive to feeding schedules. Furthermore, genetic and pharmacological impairments of the cerebellar function disrupt FAA in mice [START_REF] Mendoza | The cerebellum harbors a circadian oscillator involved in food anticipation[END_REF] . In this context, we determined the expression pattern of both PER2 and p-ERK proteins in the cerebellum of WT and GKO mice. We found no phase-adjustment of PER2 oscillations to feeding time, as well as a diminution of p-ERK expression in anticipation of feeding in GKO mice. Noteworthy, Per2 mRNA is not increased in anticipation to mealtime in Grid2 ho/ho mice that have genetic cerebellar deficits, and which do not express FAA [START_REF] Mendoza | The cerebellum harbors a circadian oscillator involved in food anticipation[END_REF] . It is thus likely that altered PER2 expression in the cerebellum of food-restricted Rev-erbα GKO mice is somewhat related to their defect to show strong food-anticipatory components. In the brain network of food-entrainable clocks, additional evidence is given here that the cerebellum may be important to integrate homeostatic signals and/or to drive FAA.

To further evaluate the central contribution of brain expression of Rev-erbα to food entrainment, we used a conditional gene KO approach. Rev-erbα expression was prevented in the nervous system by using the NesCre driver. As a result, Rev-erbα mRNA expression was barely detectable and did not cycle in the brain of BKO mice fed ad libitum or food-restricted, while Bmal1 transcript was constitutively up-regulated at the two studied time points. This however did not severely affect Per2 cycling in brain regions, in spite of reduced amplitude of Per2 transcripts between morning and evening in the SCN and cerebellum of BKO mice. In LD conditions with food provided ad libitum, brain deletion of Rev-erbα induced a more drastic reduction of both general locomotor and wheel-running activities than global Rev-erbα deletion did. Likewise, BKO mice showed a further altered circadian phenotype than that displayed by GKO mice, which is likely due to the conditional gene knockout strategy used in this study that imposes temporal and regional restriction of Rev-erbα deletion. Moreover, these changes somehow contrast with the shorter free-running period of GKO mice for Rev-erbα (our results and ref. 4), but are reminiscent of the very weak circadian rhythmicity of double GKO for Rev-erbα and Rev-erbβ [START_REF] Cho | Regulation of circadian behaviour and metabolism by REV-ERB-alpha and REV-ERB-beta[END_REF] .

Similarly to global deletion of Rev-erbα, faint expression of this gene restricted to the nervous system disrupts food entrainment. BKO mice did not show substantial FAA during the 2 weeks of food restriction. This result indicates that circadian (i.e., self-sustained) properties of FAA are altered in the absence of central Rev-erbα expression. In addition, our qPCR data in CTRL mice show that Rev-erbα expression highly responds to daytime restricted feeding in different brain nuclei, except the SCN. Furthermore, brain Rev-erbα deletion impaired changes in clock gene expression in response to limited food access in the hippocampus and cerebellum, but not in the ARC and liver. Therefore, our results indicate that Rev-erbα is essential to couple the nutritional state and the clock in some brain nuclei and support the view that the food-entrainable circadian system relies on a complex population of genes and oscillators with probably distinct roles.

Lastly, we observed that while control mice decreased their body temperature during food withdrawal, BKO animals maintained body temperature values close to those observed in conditions of food ad libitum. The absence of phase-and amplitude-adjustment of the body temperature rhythm in BKO mice may indicate that restricted feeding does not trigger adaptive mechanisms to reduce energy expenditure when food becomes scarce, to the same degree as in control mice. Interestingly, higher values of body temperature were observed not only in food-restricted GKO mice (present results), but also in 24-h food deprived or cold-exposed GKO animals [START_REF] Delezie | The nuclear receptor REV-ERBalpha is required for the daily balance of carbohydrate and lipid metabolism[END_REF][START_REF] Gerhart-Hines | The nuclear receptor Rev-erbalpha controls circadian thermogenic plasticity[END_REF] . The relative hyperthermic state of GKO and BKO could thus point to a role for Rev-erbα in the central regulation of energy expenditure. To what extent possible changes in thermoregulatory responses could have affected expression of FAA in GKO and BKO needs to be further investigated.

Together, the present findings demonstrate that Rev-erbα in the brain is required not only for proper functioning of the light-entrainable master clock, but also to integrate feeding cues and adjust circadian behaviour and physiology to environmental conditions, critical for animal survival.

Methods

Animals and housing conditions. The founders Rev-erbα heterozygous mice kindly provided by Prof. Ueli Schibler (University of Geneva, Switzerland) have been rederived on C57BL6J background [START_REF] Delezie | The nuclear receptor REV-ERBalpha is required for the daily balance of carbohydrate and lipid metabolism[END_REF] . The Rev-erbα deletion strategy is described in [START_REF] Preitner | The orphan nuclear receptor REV-ERBalpha controls circadian transcription within the positive limb of the mammalian circadian oscillator[END_REF] . NestinCre transgenic mice were obtained from The Jackson Laboratory (#003771). The conditional Rev-erbα KO model was established at the Mouse Clinical Institute (Strasbourg, France) in the framework of the European EUMODIC consortium [START_REF] Ayadi | Mouse large-scale phenotyping initiatives: overview of the European Mouse Disease Clinic (EUMODIC) and of the Wellcome Trust Sanger Institute Mouse Genetics Project[END_REF] . To create a conditional allele of Rev-erbα/Nr1d1 (MGI:2444210), an MCI proprietary vector containing flipped Neomycin resistance cassette was used, resulting in step1 plasmid. The floxed 0.6 kb fragment encompassing exons 3 and 4 was amplified by PCR and subcloned in step2 plasmid. In parallel, a 3.7 kb fragment corresponding to the 5' homology arm and 2.9 kb fragment corresponding to the 3' homology arms were amplified by PCR and subcloned in step2 plasmid to generate the final targeting construct (see Fig. S2). The linearized construct was electroporated in 129S2/SvPas mouse embryonic stem (ES) cells. After selection, targeted clones were identified by PCR using external primers and further confirmed by Southern blot with 5' and 3' external probes. Two positive ES clones were injected into C57BL/6N blastocysts, and male chimaeras derived gave germline transmission. The conditional mouse model was then backcrossed in C57BL/6J background. To produce brain-specific KO mice, we crossed animals with a conditional Rev-erbα allele with mice carrying a Cre recombinase transgene under the control of the rat Nestin promoter. To improve the efficiency of deletion of Reverbα alleles, we used the same strategy described as in ref. 37 (see Fig. S3).

Note that a significant decrease of Rev-erbα expression was detected by qPCR analysis in liver tissue of BKO mice fed ad libitum. This mild effect, however, did not affect at all the day-night expression of Bmal1 and Per2, suggesting a fully functional circadian clock in the liver (Fig. S1).

Mice of both sexes (sex ratio about 1:1) were bred in a pathogen-free facility (Chronobiotron platform, UMS 3415, CNRS and University of Strasbourg) in a temperature-controlled room (22 ± 1 °C) under 12 h light and 12 h dark (LD 12:12) conditions unless otherwise stated. Regular chow (SAFE 105, Augy, France) and water were provided ad libitum, except during the periods of restricted feeding. Access to food during restricted feeding was set automatically by the Fasting Plan system (Intellibio, Seichamps, France). Two-to 5-month old mice were individually housed in transparent plastic cages equipped with a running wheel (12.5 cm in diameter). Body mass and food intake were determined weekly. All experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals (1996), the French National Law (implementing the European Union Directive 2010/63/EU) and approved by the Regional Ethical Committee of Strasbourg for Animal Experimentation (CREMEAS).

Locomotor activity and body temperature recordings. Mice were implanted i.p. under constant gaseous anesthesia (oxygen with a constant rate at 0.2 L/min and isoflurane at 2%) with a small transponder (G2 E-Mitter, MiniMitter, Bend, Oregon, USA) recording gross locomotor activity (general activity hereafter) and core body temperature every 5 min. A PC-based acquisition system (VitalView, MiniMitter) recorded the aforementioned parameters plus the wheel-running activity 24 h a day.

Experimental design.

Global KO (GKO) -LD conditions. WT and GKO mice (n = 7-8/genotype) were fed ad libitum for 2-3 weeks and then exposed to restricted feeding schedules in which food availability was progressively reduced to 6 h per day, from ZT6 to ZT12 (ZT refers to Zeitgeber Time, ZT0 and ZT12 defining time of lights-on and lights-off, respectively) for up to 3 weeks. Of note, LD experiment was reproduced with an additional series of mice for the metabolic and mRNA/protein expression analyses presented in this study.

Global KO -DD conditions. WT and GKO mice (n = 4-6/genotype) were transferred for several weeks to constant darkness (DD) and exposed to temporal restricted feeding during which the food access was limited to 6 h per day for 3 weeks (from 02:00 p.m. to 08:00 p.m.).

Global KO -DD conditions and shift of mealtime. Mice (n = 4/genotype) were placed in DD conditions and fed ad libitum and then challenged with a restricted feeding paradigm in which food was given for 6 h from 02:00 p.m. to 08:00 p.m for 4 weeks. After a first period of food restriction at a specific circadian phase, mealtime was shifted (i.e., 6-h phase-delayed) to investigate how FAA reappears at a new feeding time.

Global KO -Skeleton photoperiod. Mice (n = 4/genotype) were exposed to skeleton photoperiod (i.e., 1 h light-pulse at the beginning of the resting period and 1 h light-pulse at the end of the resting period) and challenged with a 6-h restricted feeding schedule.

Brain-specific KO (BKO). NestinCre+ ;Rev-erbαΔ /+ (CTRL, n = 7), NestinCre+ ;Rev-erbαΔ /-(BKO, n = 7; note that one mouse was excluded a posteriori from the analysis due to poor recombination leading to normal levels of Rev-erbα in brain; see results), Rev-erbαflox/+ (n = 4) and Rev-erbαflox/-(n = 4) were fed ad libitum for 2 weeks in LD conditions and then challenged with a restricted feeding schedule in LD conditions in which food availability was reduced to 6 h per day, from ZT6 to ZT12, for up to 2 weeks. Then mice were fed ad libitum for 5 days, and were fasted for 24 h (from ZT12 to ZT12) to test the reappearance of FAA. The animals fed ad libitum were also exposed to DD conditions to evaluate their endogenous period. CTRL and BKO mice fed ad libitum were sampled in the morning (ZT0-ZT2) together with additional Rev-erbαflox/+ and Rev-erbαflox/mice fed ad libitum sampled in the evening (ZT10-ZT12) to check for recombination in the brain and liver and possible day-night variations in gene expression. Another set of CTRL and BKO mice were exposed to a restricted feeding schedule under LD for 2 weeks, as reported above. Then, brains and livers were sampled in in the morning (ZT0-ZT2) or in the evening (ZT10-ZT12).

Tissue and blood collections. GKO and WT mice from LD experiments, either with food ad libitum or restricted feeding conditions, were sacrificed at ZT0, ZT6, ZT12 and ZT18. Briefly, mice were injected with a lethal dose of pentobarbital, a blood sample was taken intracardially and two pieces of liver were cut and immediately flash-frozen in liquid nitrogen. Then animals were perfused transcardially with 50 mL PBS 1X (Phosphate-buffered saline, pH 7.2) and 50 mL PFA buffer (4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4). Brains were removed and post-fixed in 4% PFA for 24 h at 4 °C and cryoprotected successively for 24 h at 4 °C into 10, 20 and 30% sucrose solution in 0.1 M PB. Brains were then frozen in isopentane at -40 °C and then stored at -80 °C. Rev-erbΔ /-mice and their controls were sacrificed as described above. Brain were quickly removed and frozen in isopentane cooled with liquid nitrogen, and liver samples were cut and immediately flash-frozen in liquid nitrogen and stored at -80 °C.

Hormonal measurements. Plasma corticosterone was measured using an EIA kit (AC-14F1, ImmunoDiognosticSystems, Paris, France). The limit of sensitivity was 0.55 ng•mL -1 .

Immunohistochemistry. Coronal frozen brain sections (30 μ m thick) of the hypothalamus and the cerebellum of WT and GKO mice were made using a cryostat (CM3050, Leica Biosystems). Free-floating sections were rinsed in PBS 1X and incubated in a solution of 3% H 2 O 2 (Sigma-Aldrich) in PBS for 30 min at room temperature. Sections were then rinsed in PBS, and incubated for 2 h in a blocking solution containing 10% goat serum in PBS with 0.3% Tween 20 in PBS. Then sections were incubated in the primary antibody solution (in PBS + 0.3% Tween 20 + 10% goat serum) for 24 h with gentle agitation at 4 °C. We used a rabbit polyclonal anti-PER2 (1:2000; Alpha Diagnostic International, Cat. PER21-A; #869900A1) and a rabbit anti-p44/42 MAPK (1:20000; Cell Signaling #4370). Sections were then rinsed in PBS with 0.05% Tween 20 and incubated for 2 h at 4 °C with a biotinylated anti-rabbit IgG made in goat (Vectastain ABC peroxidase kit PK6101), diluted 1:500 with 0.3% Tween 20 in PBS on a plate shaker at 4 °C. Thereafter, sections were rinsed in PBS + 0.05% Tween 20 and incubated for 1 h at room temperature with an avidin-biotin-peroxidase complex (1:250; Vectastain Kit, PK6101; Vector Laboratories) in PBS + 0.05% Tween 20. Next, sections were rinsed in PBS, and incubated with 3,3′ -diaminobenzidine (0.5 mg/mL; Sigma) with 0.015% H 2 O 2 in H 2 O. Thereafter, sections were rinsed with PBS, wet-mounted onto gel-coated slides, dehydrated through a series of alcohol, soaked in toluene, and coverslipped with Eukitt (Sigma-Aldrich). Photomicrographs were taken on Leica DMRB microscope (Leica Microsystems) with an Olympus DP50 digital camera (Olympus France). The intensity and number of immunoreactive cells in hypothalamic nuclei and the Purkinje layer of the cerebellum, respectively, were determined using NIH ImageJ software (Rasband, W.S., U. S. National Institutes of Health, Bethesda MD, USA). The average intensity or cell numbers was determined, as far as possible, from three brain sections per animal. The number of animals/genotype/ZT was comprised between 3 and 6. SCN staining served as an internal control to evaluate the reliability of our immunohistochemistry experiments.

mRNA extraction and Quantitative Real-Time PCR of cerebellum and brain punches. Frozen brains from BKO mice and their controls were placed in a cryostat, cerebellum were separated from the rest of the brain, homogenized in lysis buffer supplemented with β-mercaptoethanol and total RNA was immediately extracted according to the manufacturer's protocol (RNeasy Mini Kit, Qiagen). With the remaining brain tissue, serial coronal sections (200 μ m thick) were made in the cryostat and immediately placed in RNAlater (Ambion). Brain nuclei were identified under a stereomicroscope with a mouse brain atlas and dissected with the brain punch tissue set (Leica Biosystems). Punches were put in lysis buffer and total RNA extracted using the RNeasy Micro Kit (Qiagen). RNA quantity and quality were measured using NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies) and the Bioanalyzer (Agilent RNA 6000 Pico kit, Agilent Technologies), respectively. cDNAs were synthesized from 200 ng (cerebellum) and 10-100 ng (brain punches) of total RNA using the SuperScript III Kit (Invitrogen). Quantitative Real-time PCR was performed and analyzed using an Applied Biosystems 7300 Real-time PCR System with 1X TaqMan Gene Expression Master Mix (Applied Biosystems), 1X TaqMan Gene Expression Assay (Applied Biosystems, see references below) and 1 μ L of cDNA in a total volume of 20 μ L. PCR conditions were 10 min at 95 °C followed by 40 cycles of 15 s at 95 °C, 1 min at 60 °C. PCR reactions were done in duplicate. Relative expression levels were determined using the comparative Δ C T method to normalize target gene mRNA to Tbp. The following TaqMan Gene Expression Assays were used: Bmal1 (Mm00500226_m1), Per2 (Mm00478113_m1), Nr1d1 (exon 4-5; Mm00520711_g1) and Tbp (Mm00446971_m1).

mRNA extraction and Quantitative Real-Time PCR in liver. Pieces of frozen livers from GKO mice and their control littermates, or BKO and CTRL mice were homogenized in lysis buffer supplemented with β -mercaptoethanol and total RNA was extracted according to the manufacturer's protocol (Absolutely RNA Miniprep Kit, Stratagene, Agilent technologies). RNA quantity was measured using NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies). cDNAs were synthesized from 1 μ g of total RNA using the High Capacity RNA-to-cDNA Kit (Applied Biosystems). Quantitative Real-time PCR was performed as above. Relative expression levels were determined using the comparative Δ CT method to normalize target gene mRNA to β-actin and Tbp for GKO and BKO samples, respectively. The following TaqMan Gene Expression Assays were used: β-actin (Mm01205647_g1), Bmal1 (Arnt1, Mm00500226_m1), Clock (Mm00455950_m1), Tbp (Mm00446971_m1), and Per2 (Mm00478113_m1).

Analysis of locomotor activity and temperature data. Daily rhythms of general activity and body temperature were analyzed using a Clocklab software (Actimetrics, Evanston, IL, USA) associated to MatLab (MathWorks, France). Locomotor activity data were double-plotted in actograms using the Clocklab percentile format in all the figures. Mean activity profiles were quantified every 1 h during the last 10 days of food ad libitum and restricted feeding conditions in the LD experiments while for the DD experiments only 5 days were considered in the analysis to avoid an overlap between the FAA and the activity controlled by the SCN. For comparison purpose, data points are expressed as a percentage of total daily activity. To determine the free-running period of animals in DD conditions, the χ 2 periodogram was used. Note that alpha was set at 0.01 and that 10 days were considered.

Statistical analysis.

All values are expressed as mean ± SEM. Normality and homogeneity of variance were assessed with Kolmogorov-Smirnov Lilliefors test and Levene's test, respectively. Data non-normal and/or heteroscedastic were subjected to logarithmic transformation before analysis. Alpha was set at 0.05. Data were analyzed either with Student's t-test or two-way ANOVA followed by Tukey HSD post-hoc analysis when applicable. Statistical analyses were performed with Statistica version 10 (StatSoft, Maisons-Alfort, France). For assessing daily rhythmicity of PER2 and p-ERK levels in brain structures, we used a cosinor analysis to determine mean level, amplitude and acrophase of the rhythm with SigmaPlot software (Systat software Inc., San Jose, CA, USA). Data were fitted to the following regression: [y = a + b•cos(2•π •(xc)/24)] where a is the mean level, b the amplitude, and c the acrophase of the rhythm. Cosinor regressions were considered as significant only when the 3 fitted parameters had p ≤ 0.05.

Figure 1 .

 1 Figure 1. Food-anticipatory components are reduced in food-restricted Rev-erbα GKO mice exposed to a light-dark cycle. (A) Representative double-plotted actograms (left) and thermograms (right) of a wild-type (WT) mouse (upper panel) and Rev-erbα global knock-out (GKO) mouse (lower panel) under LD conditions with food provided ad libitum and then restricted to 6 h per day (grey box, food was provided from Zeitgeber (ZT) 6 to ZT12). (B) Normalized activity profiles and body temperature raw data representing the average of 10 days during restricted feeding conditions. Daytime and nighttime are indicated by white and black bars, respectively. The period of food access during restricted feeding is represented by the grey rectangle. Data for ZT0 are double-plotted at ZT24. (C) Percent of food-anticipatory (FAA) over total daily activity from 4 h to 1 h before mealtime (left) and rise in body temperature in anticipation (right). * p < 0.05 for GKO vs. WT animals (pairwise comparisons using Tukey HSD post-hoc test after 2-way ANOVA).

Figure 3 .

 3 Figure 3. Diminished food anticipation in food-restricted Rev-erbα GKO mice exposed to constant darkness. (A) Representative double-plotted actograms (left) and thermograms (right) of a wild-type (WT) mouse (upper panels) and Rev-erbα global knock-out (GKO) mouse (lower panels) under conditions of constant darkness (DD) with food provided ad libitum and then restricted to 6 h per day (grey box, food was provided from 02:00 p.m. to 08:00 p.m.). Constant darkness is indicated by a black bar on X-axis. (B) Percent of food-anticipatory (FAA) over total daily activity (average of 5 days) from 4 h before mealtime and 2 h after mealtime (left) and rise in body temperature in anticipation (right). * p < 0.05 for GKO vs. WT animals (pairwise comparisons using Tukey HSD post-hoc test after 2-way ANOVA).

Figure 4 .

 4 Figure 4. Reduced food-anticipatory activity of Rev-erbα GKO mice in constant darkness following shift of mealtime, and in skeleton photoperiod. (A) Double-plotted actograms of wheel-running activity from two wild-type (WT) mice (upper panel) and two Rev-erbα global knock-out (GKO) mice (lower panel). Animals are in constant dark conditions (DD, indicated by the above dark bar).A few days in ad libitum (LD) feeding conditions are illustrated before transfer to DD. During restricted feeding, food access is indicated by the grey rectangle. After a first period of food restriction at a specific circadian phase, mealtime was shifted (i.e., 6-h phase-delayed). (B) Double-plotted actograms of wheel-running activity from two WT mice (upper panel) and two GKO mice (lower panel) exposed to skeleton photoperiod. Note that the 12-h light and dark phases are represented respectively by white and black bars above the actograms, and that the two 1-h light pulses are indicated by yellow lines. A few days of ad libitum (AL) feeding conditions is illustrated before food restriction (RF, grey rectangle).

Figure 5 .

 5 Figure 5. Differential changes of PER2 and p-ERK expression in the hypothalamus and cerebellum of Reverbα GKO and WT mice challenged with restricted feeding. (A-C) PER2 and (D-F) p-ERK immunoreactive (ir) cells in ventromedial (VMH) and arcuate (ARC) hypothalamic nuclei, and cerebellum (CRB) of mice under conditions of food ad libitum or exposed to restricted feeding schedules. Note that staining intensity was taken into consideration for all hypothalamic nuclei; while for the cerebellum, the number of labeled Purkinje cells was counted. a p < 0.05 for restricted feeding vs. food ad libitum at a given time-point in wild-type (WT) mice (2-way ANOVA and Tukey post-hoc test). Data for ZT0 are double-plotted.

Figure 6 .

 6 Figure 6. Clock gene mRNA levels are altered in the brain of Rev-erbα BKO mice. (A) Rev-erbα, (B) Bmal1 and (C) Per2 mRNA levels in different brain regions from Control NesCre-Rev-erbαΔ/+ (CTRL) and NesCre-Rev-erbαΔ/-(BKO) mice fed ad libitum. SCN: suprachiasmatic nucleus, ARC: arcuate nucleus, Hipp: hippocampus, CRB: cerebellum, ZT: Zeitgeber Time. * p < 0.05 for GKO vs. WT animals at a given time-point as determined by 2-way ANOVA and Tukey HSD post-hoc test; a p < 0.05 for restricted feeding vs. food ad libitum at a given time-point in wild-type (WT) mice (2-way ANOVA and Tukey post-hoc test); b p < 0.05 for restricted feeding vs. food ad libitum at a given time-point in Rev-erbα brain-specific knock-out (BKO) mice (2-way ANOVA and Tukey post-hoc test).

Figure 7 .

 7 Figure 7. Circadian activity profiles of Rev-erbα BKO mice are markedly disturbed under light-dark cycle and constant darkness. Left panels: (A) Representative double-plotted actograms of two free-fed control NesCre-Rev-erbαΔ/+ (CTRL) and (B) two NesCre-Rev-erbαΔ/-(BKO) mice under light-dark (LD) conditions and then exposed to constant darkness (DD; delimited by horizontal black arrows) for 10 days. Right panels: Corresponding χ 2 periodograms from (A) 2 CTRL and (B) 2 BKO mice.

Figure 8 .

 8 Figure 8. Food-anticipatory components are not expressed in food-restricted Rev-erbα BKO mice exposed to a light-dark cycle. (A) Representative double-plotted actograms of control NesCre-Rev-erbαΔ/+ (CTRL) animals and NesCre-Rev-erbαΔ/-(BKO) mice under LD conditions with food provided ad libitum and then restricted to 6 h per day (grey box, food was provided from ZT6 to ZT12). Note that on the last day of restricted feeding (day 24), food was given 6 h later (i.e., ZT12) than before. Thereafter, food was provided ad libitum except from day 28 (ZT12) to day 29 (ZT12) during which no food was given ("fasting test", see arrow on Y axis). (B,C, upper panels) Normalized activity profiles and body temperature raw data representing the average of 10 days during restricted feeding conditions (B) and fasting test (C) in NesCre-Rev-erbαΔ/+ (CTRL, blue circle), NesCre-Rev-erbαΔ/-(BKO, red circle), Rev-erbαflox/+ (Flox/+ , orange down-pointing triangle) and Rev-erbαflox/-(Flox/-, green up-pointing triangle) mice. Daytime and nighttime are indicated by white and black bars above the graphs, respectively. The period of food access during RF is represented by the grey rectangle. (B,C, lower panels). Statistical analysis (2-way ANOVA) was performed on percent of FAA over total daily activity and body temperature data from 4 h to 1 h before expected mealtime. a p < 0.05 for BKO vs. other groups, b p < 0.05 for BKO vs. Flox/+ and Flox/1, c p < 0.05 for CTRL vs. other groups, d p < 0.05 for Flox/+ vs. other groups, e p < 0.05 for BKO vs. Flox/+ , and f p < 0.05 for BKO vs. Flox/-.

Scientific RepoRts | 6:29386 | DOI: 10.1038/srep29386

Acknowledgements

We are indebted to Prof. Ueli Schibler (University of Geneva, Switzerland) for kindly providing the founder Rev-erbα+ /-mice. We also thank the European EUMODIC consortium for generating the Rev-erbα floxed line. We would like to thank Dr. Dominique Sage-Ciocca for management of actimetry, Magaly Mayer-Schmitt for taking part in the immunohistochemistry experiments, Sylviane Gourmelen for transponder implantation, Laurence Huck and Anita Wulgué for animal care. This work was supported by Agence Nationale pour la Recherche "Jeunes chercheurs/jeunes chercheuses" ANR-07-JCJC-0111 (E.C.), Centre National de Recherche Scientifique (E.C.), University of Strasbourg (E.C.), and a doctoral fellowship from the French Ministry of National Education and Research (J.D.).

Author Contributions

J.D. and E.C. designed the study. J.D., S.D., C.S., S.R. and P.P. carried out the research, collected the data, and contributed to data analyses. J.D. and E.C. prepared the figures and wrote the manuscript. All authors discussed the results and commented on the manuscript.

Additional Information

Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare no competing financial interests.


Adrien Moll

Romain Viennois

Martin Boehm

Michael Marek Koza

Yvan Sidis

Jérôme Rouquette

Suzon Laborde

Jérôme Debray

Bertrand Menaert

John-Paul Castellan

Anharmonicity and Effect of the Nanostructuring on the Lattice Dynamics of CrSi2

établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.


Pawan K Jha

Ewout Foppen

Andries Kalsbeek

Etienne Challet

Pawan Kumar Jha

Sleep restriction acutely impairs glucose tolerance in rats

Keywords: Glucose tolerance, sleep deprivation, suprachiasmatic nucleus, type 2 diabetes

Chronic sleep curtailment in humans has been related to impairment of glucose metabolism. To better understand the underlying mechanisms, the purpose of the present study was to investigate the effect of acute sleep deprivation on glucose tolerance in rats. A group of rats was challenged by 4h sleep deprivation in the early rest period, leading to prolonged (16 h) wakefulness. Another group of rats was allowed to sleep during the first 4 h of the light period and sleep deprived in the next 4 h. During treatment, food was withdrawn to avoid a postmeal rise in plasma glucose. An intravenous glucose tolerance test (IVGTT) was performed immediately after the sleep deprivation period. Sleep deprivation at both times of the day similarly impaired glucose tolerance and reduced the early-phase insulin responses to a glucose challenge. Basal concentrations of plasma glucose, insulin, and corticosterone remained unchanged after sleep deprivation. Throughout IVGTTs, plasma corticosterone concentrations were not different between the control and sleep-deprived group. Together, these results demonstrate that independent of time of day and sleep pressure, short sleep deprivation during the resting phase favors glucose intolerance in rats by attenuating the first-phase insulin response to a glucose load. In conclusion, this study highlights the acute adverse effects of only a short sleep restriction on glucose homeostasis.

Introduction

Recent evidence convincingly shows that sleep is important for metabolic and physiological health. Results from epidemiological studies indicate that short sleep duration for a long period is correlated with obesity and type 2 diabetes [START_REF] Gottlieb | Association of sleep time with diabetes mellitus and impaired glucose tolerance[END_REF][START_REF] Chaput | The association between sleep duration and weight gain in adults: a 6-year prospective study from the Quebec Family Study[END_REF][START_REF] Van Cauter | Sleep and the epidemic of obesity in children and adults[END_REF][START_REF] Spiegel | Effects of poor and short sleep on glucose metabolism and obesity risk[END_REF][START_REF] Watanabe | Association of short sleep duration with weight gain and obesity at 1-year follow-up: a large-scale prospective study[END_REF]. For example, habitual sleep duration of <5-6 h leads to increased body mass index and impaired glucose tolerance or even type 2 diabetes [START_REF] Vioque | Time spent watching television, sleep duration and obesity in adults living in Valencia, Spain[END_REF][START_REF] Chaput | Association of sleep duration with type 2 diabetes and impaired glucose tolerance[END_REF][START_REF] Watanabe | Association of short sleep duration with weight gain and obesity at 1-year follow-up: a large-scale prospective study[END_REF]. In addition to epidemiological studies that are mainly focused on mild chronic sleep deprivation, laboratory experiments in both human subjects and experimental animals have also linked sleep shortening with metabolic abnormalities in a more acute setting [START_REF] Spiegel | Impact of sleep debt on metabolic and endocrine function[END_REF][START_REF] Barf | Metabolic consequences of chronic sleep restriction in rats: changes in body weight regulation and energy expenditure[END_REF].

Animal experiments have shown that prolonged sleep deprivation leads to behavioral and physiological changes such as modifications in body temperature, body weight, food consumption, and energy expenditure [START_REF] Rechtschaffen | Sleep deprivation in the rat by the disk-over-water method[END_REF][START_REF] Banks | Behavioral and physiological consequences of sleep restriction[END_REF][START_REF] Nedeltcheva | Sleep curtailment is accompanied by increased intake of calories from snacks[END_REF][START_REF] Vaara | The effect of 60-h sleep deprivation on cardiovascular regulation and body temperature[END_REF][START_REF] Barf | Metabolic consequences of chronic sleep restriction in rats: changes in body weight regulation and energy expenditure[END_REF][START_REF] Markwald | Impact of insufficient sleep on total daily energy expenditure, food intake, and weight gain[END_REF]. Studies in humans have shown that the secretion of anabolic (growth hormone, prolactin, and testosterone) and catabolic hormones (glucocorticoids and catecholamines) may be affected by sleep disturbances [START_REF] Nedeltcheva | Metabolic effects of sleep disruption, links to obesity and diabetes[END_REF]. Moreover, sleep restriction lowers plasma levels of the anorexigenic hormone leptin and elevates those of the orexigenic hormone ghrelin [START_REF] Spiegel | Brief communication: Sleep curtailment in healthy young men is associated with decreased leptin levels, elevated ghrelin levels, and increased hunger and appetite[END_REF][START_REF] Taheri | Short sleep duration is associated with reduced leptin, elevated ghrelin, and increased body mass index[END_REF][START_REF] Barf | Metabolic consequences of chronic sleep restriction in rats: changes in body weight regulation and energy expenditure[END_REF]). Both quality and quantity of sleep duration may affect glucose metabolism [START_REF] Donga | A single night of partial sleep deprivation induces insulin resistance in multiple metabolic pathways in healthy subjects[END_REF][START_REF] Stamatakis | Effects of sleep fragmentation on glucose metabolism in normal subjects[END_REF][START_REF] Barf | Metabolic consequences of chronic sleep restriction in rats: changes in body weight regulation and energy expenditure[END_REF]. Furthermore, a number of experimental studies with human volunteers suggest that even partial sleep disturbance leads to impaired glucose tolerance and insulin sensitivity, that is, indicators of a prediabetic condition [START_REF] Spiegel | Impact of sleep debt on metabolic and endocrine function[END_REF][START_REF] Tasali | Slow-wave sleep and the risk of type 2 diabetes in humans[END_REF][START_REF] Donga | A single night of partial sleep deprivation induces insulin resistance in multiple metabolic pathways in healthy subjects[END_REF][START_REF] Schmid | Disturbed glucoregulatory response to food intake after moderate sleep restriction[END_REF][START_REF] Robertson | Effects of three weeks of mild sleep restriction implemented in the home environment on multiple metabolic and endocrine markers in healthy young men[END_REF]. Of note, the metabolic profile observed after sleep deprivation shares several similarities with type 2 diabetes, including decreased muscle glucose uptake, increased liver glucose output, and pancreatic bcell dysfunction [START_REF] Spiegel | Impact of sleep debt on metabolic and endocrine function[END_REF][START_REF] Buxton | Sleep restriction for 1 week reduces insulin sensitivity in healthy men[END_REF][START_REF] Donga | A single night of partial sleep deprivation induces insulin resistance in multiple metabolic pathways in healthy subjects[END_REF][START_REF] Buxton | Adverse metabolic consequences in humans of prolonged sleep restriction combined with circadian disruption[END_REF].

Most of the experiments conducted in humans and animals focused on partial or complete sleep restriction during part or the whole resting period. So far no study assessed how acute, short-term sleep deprivation affects glucose regulation. Therefore, we aimed to investigate the acute effect of short-term sleep deprivation on glucose homeostasis in rats. In order to do so, rats were kept in a light-dark cycle and transferred to constant darkness. On the first day of constant darkness animals were subjected to an intravenous glucose tolerance test (IVGTT) immediately after a 4-h sleep deprivation period in either the beginning or middle of the rest period.

Methods

All the experiments were performed in accordance with the U.S. National Institute Health Guide for the Care and Use of Laboratory Animals (1996), the French National Law (implementing the European Directive 2010/63/EU), and approved by the Regional Ethical Committee of Strasbourg for Animal experimentation (CREMEAS) and the French Ministry of Higher Education and Research (#01050.01).

Animals

Male Wistar rats (Janvier Laboratories, Le Genest-Saint-Isle, France) were maintained at 23°C under a 12-h light/ 12-h dark cycle (light intensity during light and dark periods [red light on] was 200 lux and <3 lux, respectively). Lights on at 07:00 AM and lights off at 07:00 PM defined zeitgeber time (ZT) 0 and ZT12, respectively. Animals had ad libitum access to food and water and were housed individually in Plexiglas cages (28 9 28 9 40 cm) throughout the experiments. On the day of the experiment, animals were transferred into constant darkness (DD; red light, <3 lux).

Experimental design

After a week of habituation, but only when they had reached a body weight of >300 g, animals were implanted with an intravenous silicone catheter through the right jugular vein, according to the method of [START_REF] Steffens | A method for frequent sampling of blood and continuous infusion of fluids in the rat without disturbing the animal[END_REF]. Two weeks after the surgery, when animals had gained presurgery body weight again, all animals were transferred to DD. Rats (n = 6 per group) were either sleep deprived (SD) from circadian time (CT) 0 (defining projected time of lights on during the previous light-dark cycle) to CT4 (for early subjective day sleep deprivation) or allowed to sleep from CT0 to CT4 and sleep deprived from CT4 to CT8 (for middle of subjective day sleep deprivation) by gentle handling or left undisturbed as controls (CTR). Four hours of sleep deprivation by gentle handling is enough to enhance slow-wave sleep during the recovery period in rats [START_REF] Kostin | Effect of sleep deprivation on multi-unit discharge activity of basal forebrain[END_REF]). An IVGTT was performed immediately after sleep deprivation. During the final hour of sleep deprivation the jugular vein catheter was connected to a blood sampling catheter on the top of the head. This blood sampling catheter was attached to a metal collar and guided outside the animal cage. Blood sampling catheter and metal collar were kept out of reach to the rats using a counterbalanced beam. This system allowed all manipulations to be performed outside the cage without any further handling of the animals. During the experiment (including sleep deprivation and blood sampling) no food was kept in the cages. We used red headlamp during the blood sampling in DD. A glucose solution (0.5 mL, 500 mg kg À1 body weight) was injected as a bolus via the blood sampling and jugular vein catheter. First, a blood sample (0.2 mL) was collected (t = 0), immediately followed by the glucose injection. Subsequently, blood samples (0.2 mL) were taken at t = 5, 10, 20, 40, and 60 min. Samples were used to determine plasma concentrations of glucose, insulin, and corticosterone at these time points. The total amount of glucose in plasma and total amount of insulin released after the glucose bolus injection was calculated from the area under the curve (AUC) of every individual animal and averaged for the experimental groups.

Laboratory method/analysis

During the experiment, blood glucose concentrations were determined by a glucometer (Accu-Check, Roche Diagnostic, Meylan, France). Blood samples were collected in tubes on ice containing heparin and later centrifuged at +4°C. Plasma was isolated and stored at À20°C for further analysis of insulin and corticosterone. Plasma concentrations of insulin and corticosterone were measured employing radioimmunoassay kits (Millipore, Billerica for insulin and MP Biomedicals, Orangeburg for corticosterone).

Statistical analysis

Data are presented as mean AE standard error of the mean (SEM). Statistical analysis was performed by SigmaPlot (version 12, SPSS Inc., Chicago, IL). Significance was defined at P < 0.05. Two-way analysis of variance (ANO-VAs) with repeated measures (rmANOVA) were performed to compare glucose, insulin, and corticosterone levels for different samples. Three-way ANOVAs were performed to compare glucose, insulin, and corticosterone levels according to sample timing and sleep status at the two CTs. Twoway ANOVAs were performed to compare basal glucose, insulin, corticosterone, AUCs, I/G 5-0 , and I/G 10-5 between the experimental groups at the two CTs. If appropriate, post hoc analysis was performed using Tukey's test.

Results

Intravenous glucose tolerance test were performed immediately after the sleep deprivation (SD), during the beginning as well as in the middle of the subjective day. SD in both early and midsubjective day caused an impaired glucose tolerance. Injection of the glucose bolus resulted in an immediate and pronounced increase in plasma concentrations of glucose and insulin in both control and sleep-deprived animals (Fig. 1A,B,D andE). Highest glucose concentrations were detected 5 min after the bolus injection, directly followed by a rapid decrease. Within 20 min after injection, glucose concentrations had returned to preinfusion concentrations again. Both during early and midsubjective day, ANOVA showed significant effects of SD (F 1,50 = 6.42, P = 0.03 and F 1,50 = 13.42, P = 0.004), sample timing (F 5,50 = 34.85, P < 0.001 and F 5,50 = 35.63, P < 0.001), and interaction (F 5,50 = 5.56, P < 0.001 and F 5,50 = 12.31, P < 0.001). Post hoc analysis revealed that plasma glucose levels were significantly elevated at t=5 min in sleep-deprived compared to control animals at both CT4 and CT8 (P < 0.001). The three-way ANOVA showed no significant effects of time of day (P = 0.194) or the interaction of sample timing 9 time of day (P = 0.885), SD 9 time of day (P = 0.512), or sample timing 9 SD 9 time of day (P = 0.587), indicating that the glucose responses at both time points were very similar.

Plasma insulin levels also increased in response to the glucose bolus in both the sleep-deprived and control groups, at t = 5 min. ANOVA showed no significant effect of SD during either early or midsubjective day (F 1,50 = 0.73, P = 0.41 and F 1,50 = 0.25, P = 0.62), but sample timing (F 5,50 = 18.72, P < 0.001 and F 5,50 = 2.44, P = 0.046) and interaction (F 5,50 = 2.89, P = 0.02 and F 5,50 = 4.46, P = 0.002) did show significant effects at both time points. Post hoc analysis revealed that insulin levels were significantly higher in the sleep-deprived group at t = 10 min during both the beginning and the middle of the rest period (P = 0.025 and P = 0.004). In addition, at t = 40 min plasma insulin levels were increased in the control group (CT4-8) (P = 0.02). The three-way ANOVA showed significant effects of sample timing 9 time of day (P = 0.035) and sample timing 9 SD 9 time of day (P = 0.012), but not time of day (P = 0.838) or SD 9 time of day (P = 0.392) interactions, indicating small time-course differences in insulin responses during IVGTT at CT4 and CT8 (see Fig. 1B andE).

To test the possibility of activation of the hypothalamo-pituitary-adrenal (HPA) axis due to sleep deprivation and intervention of IVGTTs, we measured corticosterone levels before and during IVGTTs. Basal levels of plasma corticosterone were not affected by sleep deprivation (F 1,20 = 0.03, P = 0.8), but basal levels were higher at CT8 than at CT4 (F 1,20 = 9.1, P = 0.007) (Fig. 2C). During IVGTTs, ANOVA showed no significant effect of SD during either early or midsubjective day (F 1,50 = 0.67, P = 0.43 and F 1,50 = 0.38, P = 0.54) (Fig. 1C andF). The three-way ANOVA did not show significant effects of SD 9 time of day (P = 0.99), SD 9 sample timing (P = 0.92) and sample timing 9 SD 9 time of day (P = 0.9), but it detected an effect of time of day (P = 0.01) and sample timing 9 time of day (P = 0.04), indicating the higher mean corticosterone levels during the CT4-8 IVGTT.

Basal levels of plasma glucose did not change due to sleep deprivation (F 1,20 = 2.54, P = 0.12), though the effect of time of day on plasma glucose concentration was apparent (F 1,20 = 10.33, P = 0.004), with higher levels later in the day (Fig. 2A). Like basal glucose, basal plasma concentrations of insulin also depended on the time of day (F 1,20 = 7.55, P = 0.012) (Fig. 2B). Post hoc analysis showed that especially in the SD group, basal insulin was higher at CT8 compared to CT4 (P = 0.017).

To estimate the ability of the b cells to respond to a glucose challenge, we calculated insulin secretion over the first 5 min after the injection (ΔI5-0) divided by the difference between the glucose concentrations during the same time period (ΔG5-0), that is, I/G 5-0 . ANOVA showed significant effects of SD (F 1,20 = 12.91, P = 0.002) and time of day (F 1,20 = 6.40, P = 0.02) (Fig. 2D, Table 1), with I/G 5-0 being lower at CT8. Post hoc analysis revealed that SD significantly decreased the I/G 5-0 in both early and late subjective day (P = 0.008 and P = 0.044).

We further tested the ability of the b cells to respond to a glucose load at 10 min after injection. For this, we calculated the I/G 10-5 . ANOVA showed significant effects of SD (F 1,20 = 10.8, P = 0.004), but not time of day (F 1,20 = 1.2, P = 0.27) or its interaction (F 1,20 = 0.48, P = 0.49) (Table 1). Post hoc analysis revealed that SD significantly decreased the I/G 10-5 only during the early subjective day (P = 0.01).

We also analyzed the AUCs as estimation for the amount of glucose and insulin released after the bolus injection of glucose (Fig. 2E andF). ANOVA showed that SD significantly affected the AUC of glucose (F 1,20 = 17.77, P < 0.001). Post hoc analysis revealed that SD significantly increased the glucose AUC in early and middle of subjective day (P = 0.01 and P = 0.006). On the other hand, insulin AUCs were not significantly affected by SD (F 1,20 = 1.18, P = 0.29) and time of day (F 1,20 = 0.64, P = 0.43).

Discussion

There is increasing evidence from human and animal studies that disturbed sleep is associated with perturbations in glucose homeostasis [START_REF] Spiegel | Impact of sleep debt on metabolic and endocrine function[END_REF][START_REF] Barf | Chronic sleep disturbance impairs glucose homeostasis in rats[END_REF]. It is not clear, however, how acute sleep deprivation in terms of duration and timing during the rest period impacts on glucose metabolism. In the present study, we show in rats that a short period (4 h) of sleep deprivation is sufficient to impair glucose tolerance and reduce the early-phase insulin response to an intravenous glucose load.

Methodological considerations

The detrimental impact on glucose metabolism of short sleep duration over many days together with misaligned or irregular sleep has been reported in several studies For sleep deprivation during early daytime, rats were forced to be awake during the first 4 h of the usual resting period (CT0-4), thus prolonging the period of wakefulness from about 12 h to 16 h. For sleep deprivation later during the light period, there are two options: either keeping the rats awake during a longer time span (e.g., 20 h) or allowing sleep during the early part of the rest period followed by sleep restriction during the latter part of the rest period. We chose the latter option as it permits to test the effect of a similar period of sleep deprivation (i.e., 4 h), but occurring at a different time of day. Notwithstanding, the fact that with sleep deprivation during the latter part of the light period sleep propensity was probably decreased compared to the rats sleep deprived in early morning, glucose tolerance was similarly altered in rats sleep deprived in either the early or middle part of the rest period. Thus, independent of time of day and sleep pressure, sleep restriction is capable of altering glucose homeostasis. In fact, the adverse effect of sleep deprivation on glucose tolerance was much stronger than the diurnal variation in glucose tolerance. Thus, the effect of sleep deprivation completely overruled the improvement of glucose tolerance during the light period as seen in the control animals.

In human studies that investigated the effects of acute sleep restriction [START_REF] Schmid | Mild sleep restriction acutely reduces plasma glucagon levels in healthy men[END_REF][START_REF] Donga | A single night of partial sleep deprivation induces insulin resistance in multiple metabolic pathways in healthy subjects[END_REF], lights were on during sleep restriction, which could stimulate wakefulness and inhibit melatonin secretion [START_REF] Redlin | Neural basis and biological function of masking by light in mammals: suppression of melatonin and locomotor activity[END_REF][START_REF] Chellappa | Can light make us bright? Effects of light on cognition and sleep[END_REF]. In rats light exposure has been reported to stimulate glucocorticoid release [START_REF] Buijs | Anatomical and functional demonstration of a multisynaptic suprachiasmatic nucleus adrenal (cortex) pathway[END_REF]) and increase plasma glucose [START_REF] Challet | Daily variations of blood glucose, acid-base state and PCO2 in rats: effect of light exposure[END_REF]). Thus, to avoid interferences with the outcomes studied, lights were turned off during the present experiment. Moreover, to rule out any putative bias due to changes in food intake of sleep-deprived rats, food was removed before the start of sleep deprivation and during blood sampling.

Several procedures have previously been used to induce sleep deprivation in rodents, including forced locomotion, gentle handling, and short platform over water. Gentle handling has the advantage to avoid the confounding effect of hyperactivity triggered by forced locomotion. In addition, it is thought to prevent the stressful effects of platform over water and forced locomotion. Our assumption that gentle handling is suitable for short periods of sleep deprivation is supported by finding similar levels of basal blood corticosterone in control and sleep-deprived rats, indicating that the experimental groups were not stressed by gentle handling.

Glucose tolerance and hormonal changes

In our study, a single period of 4 h of sleep deprivation either in early or middle of the light period did not modify the basal levels of plasma glucose, insulin, and corticosterone, a finding consistent with the lack of significant effect of a single night limited to 4.5 h sleep in human subjects [START_REF] Schmid | Mild sleep restriction acutely reduces plasma glucagon levels in healthy men[END_REF]. The data from the IVGTT show that sleep deprivation in rats strongly reduces glucose tolerance, as evidenced by the rise in plasma glucose concentrations to higher levels and for a longer time. Several effects may participate in the reduced glucose tolerance. First, although the total amount of insulin released in the sleep-deprived group was not changed (Fig. 2F), the reduced early insulin responses at both time points investigated indicates a reduced or at least inadequate sensitivity of the b cells. During the first 5 min after injection of the glucose bolus insulin levels were similar in the control and sleep-deprived group, but the responsiveness of b cells to the glucose load was significantly reduced in the sleep-deprived group at CT4 and CT8, and at CT4 this effect even remained present in the next 5 min. These findings are very similar to those of a previous rat study using a much longer period of sleep deprivation (i.e., 20 h) [START_REF] Barf | Chronic sleep disturbance impairs glucose homeostasis in rats[END_REF].

The decreased glucose tolerance in sleep-deprived animals during IVGTT may results from either higher glucose production or less glucose uptake. The data from the present study could not differentiate whether the hyperglycemia is due to reduced glucose uptake or more glucose production. The reduced early insulin response in the sleep-deprived groups will result both in a reduced glucose uptake as well as a lesser inhibition of glucose production. In order to understand further the mechanism of hyperglycemia, experiments using the stable isotope dilution technique to determine endogenous glucose production need to be done. Sleep deprivation might trigger glucagon release, which would subsequently result in a higher endogenous glucose production. Although at a first glance, this hypothesis appears unlikely because acute sleep deprivation has an inhibitory effect on circulating glucagon levels in humans [START_REF] Schmid | Mild sleep restriction acutely reduces plasma glucagon levels in healthy men[END_REF], further 2016 | Vol. 4 | Iss. 12 | e12839 assays of plasma glucagon are needed to evaluate this possibility.

An alternative explanation for the increased glucose levels during the IVGTT could be an increased activity of the HPA axis, as a consequence of stress during acute sleep deprivation. In humans, most studies reported no acute change in glucocorticoid levels after sleep deprivation [START_REF] Everson | Reductions in circulating anabolic hormones induced by sustained sleep deprivation in rats[END_REF][START_REF] Donga | A single night of partial sleep deprivation induces insulin resistance in multiple metabolic pathways in healthy subjects[END_REF]), although delayed effects (i.e., the day after) have been reported [START_REF] Leproult | Sleep loss results in an elevation of cortisol levels the next evening[END_REF]. By contrast, depending on the procedure of sleep deprivation in animal studies, sleep disturbances can increase glucocorticoid release [START_REF] Baud | Sustained sleep fragmentation affects brain temperature, food intake and glucose tolerance in mice[END_REF]). However, no differences were reported in plasma corticosterone levels between sleep-deprived and control rats [START_REF] Barf | Chronic sleep disturbance impairs glucose homeostasis in rats[END_REF]). In the present study, basal levels of plasma corticosterone and corticosterone release during IVGTTs were not different in sleepdeprived rats as compared to undisturbed controls, ruling out the possibility of major acute activation of the adrenal via the HPA or sympatho-adrenal axis.

Possible mechanisms

Our results revealed an altered insulin response to the glucose load during the first 5 min in sleep-deprived animals. The diminished early-phase response of insulin after sleep deprivation suggests a reduced or impaired sensitivity of the b cells to a glucose challenge. This defect may depend on disturbances in the sensitivity of the pancreatic b cells to glucose and/or its control by the autonomic nervous system. The latter possibility is supported by the fact that sleep deprivation results in sympathetic activation and release of catecholamines in the general circulation [START_REF] Levy | Sleep, sleepdisordered breathing and metabolic consequences[END_REF]. Hyperactivity of the sympathetic branch of the autonomic nervous system may lead to insulin resistance [START_REF] Egan | Insulin resistance and the sympathetic nervous system[END_REF]. Thus, the reduction in the early-phase insulin response to glucose might be related to an increased sympathetic and/or decreased parasympathetic activity. Moreover, increased activity of the sympathetic nervous system would also stimulate glucose production. Future work should determine possible changes in the sympathovagal balance under the present conditions of sleep deprivation.

Considering that some actions of sleep deprivation on peripheral functions may result from sympathetic activation, what could be the central structures mediating these effects? A likely candidate is the hypothalamic orexin system, because this neuropeptide is involved not only in the regulation of the sleep/wake cycle, but also in the daily rhythm of glucose metabolism [START_REF] Sakurai | The neural circuit of orexin (hypocretin): maintaining sleep and wakefulness[END_REF]Kalsbeek et al. 2010b). Activity of orexin neurons in the perifornical region of the hypothalamus is highest during the wake period and during sleep deprivation [START_REF] Estabrooke | Fos expression in orexin neurons varies with behavioral state[END_REF]. These orexin neurons also participate in the control of endogenous glucose production in the liver via the autonomic nervous system [START_REF] Yi | A major role for perifornical orexin neurons in the control of glucose metabolism in rats[END_REF]). Furthermore, orexin appears to regulate insulin sensitivity, because mice lacking orexin show an age-related development of systemic insulin resistance [START_REF] Hara | Difference in obesity phenotype between orexin-knockout mice and orexin neuron-deficient mice with same genetic background and environmental conditions[END_REF][START_REF] Tsuneki | Age-related insulin resistance in hypothalamus and peripheral tissues of orexin knockout mice[END_REF]. Finally, orexin has bidirectional effects on hepatic gluconeogenesis via the autonomic nervous system [START_REF] Tsuneki | Hypothalamic orexin prevents hepatic insulin resistance via daily bidirectional regulation of autonomic nervous system in mice[END_REF]. To test whether orexin neurons are involved in the autonomic control of hepatic glucose production and/or pancreatic sensitivity to glucose, orexin antagonist and organ-specific denervation studies should be performed during sleep deprivation.

Like orexin, also the serotonin system is involved in arousal and the regulation of glucose metabolism [START_REF] Asikainen | Sleep deprivation increases brain serotonin turnover in the rat[END_REF][START_REF] Versteeg | Serotonin, a possible intermediate between disturbed circadian rhythms and metabolic disease[END_REF]. Injection of serotonin leads to hypoglycemia in rats and mice [START_REF] Yamada | Serotonin-induced hypoglycemia and increased serum insulin levels in mice[END_REF][START_REF] Sugimoto | Effects of serotonin on blood glucose and insulin levels of glucose-and streptozotocin-treated mice[END_REF]. Mice deficient in serotonin reuptake transporters and the 5-HT 2c receptor in pro-opiomelanocortin neurons of the arcuate nucleus in the hypothalamus show impaired glucose metabolism [START_REF] Xu | 5-HT2CRs expressed by pro-opiomelanocortin neurons regulate insulin sensitivity in liver[END_REF][START_REF] Chen | Reduced serotonin reuptake transporter (SERT) function causes insulin resistance and hepatic steatosis independent of food intake[END_REF]. The daily rhythm of SCN serotonin was shown to be severely impaired in glucose intolerant hamsters, indicating a functional link between the SCN, serotonin, and glucose metabolism [START_REF] Luo | Suprachiasmatic nuclei monoamine metabolism of glucose tolerant versus intolerant hamsters[END_REF]). However, additional experiments are needed before the hypothalamic serotonin system can firmly be implicated in the sleep deprivation-induced changes in glucose metabolism.

NPY is another hypothalamic neuropeptide involved in the control of feeding, arousal, and glucose metabolism [START_REF] Szentirmai | Central administration of neuropeptide Y induces wakefulness in rats[END_REF]Kalsbeek et al. 2010a;[START_REF] Wiater | Circadian integration of sleep-wake and feeding requires NPY receptorexpressing neurons in the mediobasal hypothalamus[END_REF]. Chronic sleep deprivation studies have shown increased expression of hypothalamic NPY [START_REF] Koban | Changes in hypothalamic corticotropin-releasing hormone, neuropeptide Y, and proopiomelanocortin gene expression during chronic rapid eye movement sleep deprivation of rats[END_REF][START_REF] Martins | Orexin activation precedes increased NPY expression, hyperphagia, and metabolic changes in response to sleep deprivation[END_REF]. Central administration of NPY results in an increase in EGP in rats, probably by increasing hepatic glucose production (Kalsbeek et al. 2010a). The i.c.v. administration of NPY causes insulin resistance via activation of sympathetic output to the liver (van den [START_REF] Van Den Hoek | Intracerebroventricular administration of neuropeptide Y induces hepatic insulin resistance via sympathetic innervation[END_REF]. NPY-containing neurons in the arcuate nucleus also project to the paraventricular nucleus of the hypothalamus (PVN), which is a relay center for the hypothalamic integration of glucose metabolism. Therefore, the presently observed impaired glucose tolerance might have been mediated through an enhanced stimulation of NPY receptors in the hypothalamus.

Biomedical perspectives

The present study investigated the acute effects of sleep deprivation on glucose homeostasis in rats. Our data show that disturbance of the sleep-wake rhythm during early or late subjective day by short sleep deprivation acutely affects glucose metabolism by impairing glucose tolerance. Our results show that prolonged wakefulness (sleep deprivation during the early resting period) and short duration sleep deprivation (sleep deprivation in the middle of the rest period) impair glucose tolerance to the same extent.

The sleep-wake cycle is oppositely phased in nocturnal and diurnal species according to the astronomical light/ dark cycle, while plasma glucose concentrations also show oppositely phased rhythms between nocturnal and diurnal rodents [START_REF] Dardente | Daily and circadian expression of neuropeptides in the suprachiasmatic nuclei of nocturnal and diurnal rodents[END_REF]). Therefore, it would be interesting to determine whether acute sleep deprivation during the resting period induces the same alterations of glucose metabolism in a diurnal rodent, that is, being active during the light period as are humans.

Unraveling the mechanisms that underlie the deleterious effects of sleep deprivation on glucose metabolism in rodents under tightly controlled conditions may be ultimately relevant for applications in humans.

Figure 1 .

 1 Figure 1. Intravenous glucose tolerance tests (IVGTTs) in rats after sleep deprivation. Relative changes in plasma glucose concentration (A, D), plasma insulin concentration (B, E), and plasma corticosterone concentration (C, F) after a glucose bolus (500 mg kg À1 intravenous) during IVGTTs starting at circadian time (CT) 4 and CT8. Black circles: control animals; gray squares: sleep-deprived animals. CTR = control, SD = sleep deprived. All groups n = 6. Data are presented as mean AE SEM. *P < 0.05, **P < 0.005, ***P < 0.001.

Figure 2 .

 2 Figure 2. Basal glucose and hormone concentrations during intravenous glucose tolerance tests (IVGTTs) in rats after sleep deprivation (all groups n = 6). (A) Basal plasma glucose concentrations were significantly higher at circadian time (CT) 8 compared to CT4. (B) Basal plasma insulin concentrations were significantly higher at CT8 in the SD group. (C) Basal plasma corticosterone concentrations were significantly higher at CT8 compared to CT4. (D) I/G t0-5 : the ratio of ΔI5-0 to ΔG5-0 (ΔI5-0/ΔG5-0 as a measure of the insulin response to glucose in the first 5 min) was reduced significantly after sleep deprivation in the early and late rest period. (E) AUC of the plasma glucose response and (F) the plasma insulin response after a glucose bolus at CT4 and CT8 after a 4-h sleep deprivation. Black bars: control group; gray bars: sleep-deprived group. CTR = control, SD = sleep deprived, AUC = area under the curve. AUC was calculated from t = 0 till t = 20 min. Data are presented as mean AE SEM. *P < 0.05 between CTR and SD; ## P < 0.005, # P < 0.05 between CT4 and CT8.

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  2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society. 2016 | Vol. 4 | Iss. 12 | e12839

Table 1 .

 1 b cells responsiveness to glucose load expressed as I/G.[START_REF] Briancon-Marjollet | The impact of sleep disorders on glucose metabolism: endocrine and molecular mechanisms[END_REF]. A few studies also investigated the effects of acute sleep restriction (i.e., within one circadian cycle) on glucose homeostasis in humans[START_REF] Schmid | Mild sleep restriction acutely reduces plasma glucagon levels in healthy men[END_REF][START_REF] Donga | A single night of partial sleep deprivation induces insulin resistance in multiple metabolic pathways in healthy subjects[END_REF]) and rats[START_REF] Barf | Chronic sleep disturbance impairs glucose homeostasis in rats[END_REF]. In both cases, the effects of sleep restriction were tested at only one time point. In both humans and rats glucose homeostasis is strongly influenced by time of day[START_REF] Kumar Jha | Circadian rhythms in glucose and lipid metabolism in nocturnal and diurnal mammals[END_REF]. Among others, glucose tolerance improves from the beginning of the rest period to the onset of the activity onset(la Fleur et al. 2001). Such daily variations may thus modulate the effects of sleep deprivation on glucose metabolism. Therefore, we set out to investigate whether the effects of sleep deprivation are influenced by time of day.

	CT4

CTR = control, SD = sleep deprived. Data are presented as mean AE SEM (n = 6/group). * P < 0.05 between CTR and SD. ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society. 2016 | Vol. 4 | Iss. 12 | e12839 (Briancon-

ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

Acknowledgments

We thank Dr. Hanan Bouaouda and Sylviane Gourmelen for their assistance in SD procedure and animal care, respectively. We are also indebted to Pr. Paul P evet for continuous support.

Funding Information

This work was supported by a doctoral fellowship from the "Neurotime" Erasmus Mundus program, European Doctoral College of University of Strasbourg and Euromotropolis of Strasbourg (PKJ), and grants from Centre National de la Recherche Scientifique (EC), University of Strasbourg (EC), and University of Amsterdam (AK).

Conflict of Interest

None declared.