Recent studies have shown that a high‐fat (HF) diet leads to rapid changes in both the period of locomotor activity in constant darkness and to increased food intake during the normal rest period under light‐dark conditions ( 21 ). We and others have shown that these changes in behavioral rhythmicity correlate with disrupted circadian gene expression within hypothalamus, liver, and adipose tissue and with altered cycling of hormones and nuclear hormone receptors involved in fuel utilization, such as leptin, thyroid‐stimulating hormone, and testosterone in mice, rats, and humans ( 21 - 26 ). Although nocturnal mice fed an HF diet during the whole of the light phase gained significantly more weight than mice fed during the dark period ( 27 , 28 ), an HF diet has never been tested for its effect under RF. Our aim in this study was to determine whether combination of the clock‐resetting feeding regimen (daytime RF) can attenuate the disruptive effect that diet‐induced obesity has on circadian expression of metabolic factors.

Restricted feeding (RF) limits the time and duration of food availability without calorie reduction ( 17 ), i.e ., food is provided ad libitum (AL) for ∼3–5 h at the same time every day, usually during daytime. Interestingly, daytime RF affects the circadian clocks in peripheral tissues, such as liver, kidney, heart, and pancreas, causing uncoupling from the central pacemaker in the SCN. Many physiological activities normally dictated by the SCN are altered by daytime RF ( 18 - 20 ). We have recently shown that long‐term daytime RF can increase the amplitude of clock gene expression, increase expression of catabolic factors, and reduce the levels of disease markers, leading to better health ( 17 ).

Disrupted circadian rhythms lead to attenuated circadian feeding rhythms, hyperphagia, diabetes, and obesity ( 5 - 8 ) because the circadian clock regulates the expression and/or activity of certain metabolic enzymes, hormones, and transport systems ( 5 ). In addition, the core clock mechanism is tightly linked to metabolic pathways, as follows. Reverse erythroblastosis virus α (REV‐ERBα), retinoic acid receptor‐related orphan receptor α (RORα), and peroxisome proliferator‐activated receptor α (PPARα), regulators of lipogenesis and lipid metabolism, regulate Bmal1 transcription ( 9 - 11 ). In turn, the CLOCK:BMAL1 heterodimer regulates the expression of Rev‐erb α, Ror α, and Ppar α (7, 9–12). Activation of adenosine monophosphate‐activated protein kinase (AMPK), a sensor of low energy and nutrient state in the cell, leads to altered circadian rhythms by destabilizing the negative limb of the circadian clock, PERs, and CRYs ( 13 , 14 ). Sirtuin 1 ( SIRT1 ), a key factor involved in metabolism, interacts directly with CLOCK and deacetylates BMAL1 and PER2 ( 15 , 16 ).

Mammalian homeostatic systems have adapted to daily changes in light and dark by developing an endogenous circadian clock, located in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus, which generates endogenous rhythms of ∼24 h ( 1 ). Similar clocks are found in peripheral tissues, such as the liver, intestine, and adipose tissue ( 2 - 4 ). The clock mechanism in the brain and peripheral tissues consists of CLOCK and BMAL1 that heterodimerize and bind to E‐box sequences to mediate transcription of a large number of genes, including Periods ( Pers ) and Cryptochromes ( Crys ). PERs and CRYs constitute part of the negative feedback loop and inhibit CLOCK:BMAL1‐mediated transcription ( 1 ).

All results are expressed as means ± se. One‐way ANOVA (time of day) was performed to analyze the circadian pattern of clock and metabolic genes and proteins with several time points. Tukey's honestly significant difference (HSD) was performed as a single‐step multiple comparison procedure and statistical test in conjunction with ANOVA for the evaluation of significant differences among the groups in average daily expression levels of metabolic genes and proteins. For all analyses, the significance level was set at P < 0.05. Statistical analysis was performed with JMP 5.1 software (SAS Institute, Inc., Cary, NC, USA). Further analysis of circadian rhythmicity was performed using Acro 3.5 software (Circadian Rhythm Laboratory, University of South Carolina, Walterboro, SC, USA).

Liver samples (∼200 mg) were homogenized in lysis buffer, as was described previously ( 30 ). Samples were run onto a 10% SDS‐polyacrylamide gel [for AMPK, phosphorylated (p) AMPK, SIRT1, acetyl coenzyme A carboxylase (ACC), and pACC]. After electrophoresis, proteins were semidry‐transferred onto nitrocellulose membranes. Blots were incubated with anti‐mouse AMPK/pAMPK and ACC/pACC polyclonal antibody (Cell Signaling Technology, Beverly, MA, USA) and with anti‐mouse SIRT1 polyclonal antibody (Abcam, Cambridge, UK). Anti‐mouse actin antibody (MP Biomedicals, Solon, OH, USA) was used to detect actin, the loading control. Membranes were washed and reacted with horseradish peroxidase‐conjugated anti‐goat (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) and anti‐mouse (Jackson ImmunoResearch, West Grove, PA, USA) antibodies. The immune reaction was detected by enhanced chemiluminescence (Santa Cruz Biotechnologies). Finally, bands were quantified by scanning and densitometry; results are expressed as arbitrary units.

RNA was extracted from liver using TRI Reagent (Sigma, Rehovot, Israel). Total RNA was DNase I‐treated using RQ1 DNase (Promega, Madison, WI, USA) and reverse‐transcribed using MMuLV reverse transcriptase and random hexamers (Promega), as was described previously ( 17 ). The reaction was subjected to quantitative real‐time PCR using primers spanning exon‐exon boundaries and the ABI Prism 7300 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Primers ( Table 1 ) for all genes were tested alongside the normalizing gene Gapdh . The fold change in target gene expression was calculated by the 2 −ΔΔ C t relative quantification method (Applied Biosystems).

General cage activity was monitored continuously at 6‐min intervals using a custom‐made system composed of infrared detectors placed above each cage, as was described previously ( 29 ). Cage locomotor activity was recorded continuously under LD conditions, after which mice were released into DD for 10 d, and activity recording was continued. Doubleplotted actograms were generated using Actogram software (kindly provided by Roberto Refinetti, Circadian Rhythm Laboratory, University of South Carolina. Walterboro, SC, USA) Period lengths of circadian activity rhythms in DD ( tau ) were calculated individually by χ 2 analyses using Tau software (kindly provided by Roberto Refinetti).

Blood was kept at room temperature for 30 min for clotting and consequently centrifuged at 2000 g for 15 min. Serum was collected and stored at —80°C for further analysis. Serum hormone levels were determined for insulin (Mercodia, Uppsala, Sweden), adiponectin (Millipore Corp., Billerica, MA, USA), ghrelin (Linco Research, St. Charles, MO, USA), corticosterone (Assaypro, St. Charles, MO, USA), leptin, IL‐6, and TNF‐α (R&D Systems, Inc., Minneapolis, MN, USA) using ELISA kits. Assays were performed according to the manufacturers' instructions.

Four‐week‐old C57BL/6 male mice were housed in a temperature‐ and humidity‐controlled facility (23–24°C, 60% humidity). Mice were entrained to a light‐dark (LD) cycle of 12 h of light and 12 h of darkness for 2 wk with food available ad libitum (AL). After 2 wk, mice were fed AL or RF, and each group was divided into 2 subgroups that were fed either a HF diet or a low‐fat (LF) diet for 18 wk. The HF diet was based on soybean oil and palm stearin (fatty acid composition: C:12, 0.3%; C:14, 1.3%; C:16, 55%; C:18, 5.1%; C:18–1, 29.5%; C:18–2, 7.4%; and C:18–3, 0.7%) and contained 22% w/w fat (42% kcal from fat) vs . soybean oil 7% w/w (16% kcal from fat) for the LF diet. The RF group was given food between zeitgeber time 4 and 8 (zeitgeber time 0 is the time of lights on). Daily food intake and body weight were monitored once weekly throughout the experiment. Mice were anesthetized with isoflurane, and blood and liver samples were removed every 4 h around the circadian cycle in total darkness [dark‐dark (DD) cycle] under a dim red light to avoid the masking effects by light. Fasting blood glucose levels were determined using a glucometer (Optium Xceed; Abbott Laboratories, Maidenhead, UK). Tissues were immediately frozen in liquid nitrogen and stored at —80°C until further analysis. Mice were humanely killed at the end of the experiment. The joint ethics committee (institutional animal care and use of committee) of the Hebrew University and Hadassah Medical Center approved this study.

We next measured the key metabolic factors AMPK, SIRT1, ACC, and PPARα ( Fig. 5 ), whose activation leads to induction of catabolic pathways and that have recently been shown to play an important role in the core clock mechanism ( 5 , 31 ). As expected, the RF‐LF diet led to increased levels of pAMPK (Fig. 5B ) and pACC (Fig. 5E ; P <0.05, Tukey's HSD), indicating intracellular low energy levels, inhibition of fatty acid synthesis and increased fatty acid oxidation. The AL‐HF diet down‐regulated AMPK (Fig. 5B ), ACC (Fig. 5D ), and SIRT1 (Fig. 5G ) daily protein levels by ∼50% compared with AL‐LF mice. Interestingly, the timed HF diet led to 37% lower levels of pAMPK (Fig. 5C ) than those in the RF‐LF group and 62% increased pACC levels (Fig. 5E ) compared with the AL‐LF group, indicating adequate energy levels but reduced fatty acid synthesis. In addition, RF‐HF mice exhibited increased daily levels of Ppar α mRNA (Fig. 5H ). Similar to the effects on clock gene oscillation, the HF diet led to disruptions and phase delays in expression of Ampk and Sirt1 mRNA and pAMPK and ACC protein, whereas the RF‐HF diet led to a phase advance in the expression of Ampk, Sirt1 , and Ppar α mRNA and pAMPK protein ( P <0.05, 1‐way ANOVA; data not shown). These results demonstrate again the dominance of the timed feeding over the HF diet.

Circadian mRNA expression of liver clock genes of mice fed the AL‐LF, AL‐HF, RF‐LF, and RF‐HF diet. A ) Per1 mRNA. B ) Per2 mRNA. C ) Bmal1 mRNA. D ) Cry1 mRNA. E ) Cry2 mRNA. F ) Clock mRNA. G ) Rev‐erb α mRNA. H ) Ror α mRNA. I ) CkI∊ , mRNA. Tissues were collected every 4 h around the circadian cycle. Total RNA was extracted, and real‐time PCR analyses were performed to determine mRNA levels. Data are means ± se; n = 6 for each time point in each group. Shaded and solid bars designate the subjective day and night, respectively; cross‐hatched bar indicates food availability during RF.

We next measured the effect of the timed HF diet on clock gene expression in the periphery. Our analyses revealed that liver Per1, Per2, Bmal1, Cry1, Cry2 ( Fig. 4A–E ), and Rev‐erb α (Fig. 4G ) oscillated robustly in all groups ( P <0.05, 1‐way ANOVA). Clock (Fig. 4F ) and Ror α (Fig. 4H ) oscillated robustly in all diet groups except for RF‐LF, and CkI∊ oscillated robustly only in the LF groups (Fig. 4I ). The AL‐HF diet disrupted the circadian expression, causing a phase advance of Clock (Fig. 4F ) and Per1 (Fig. 4A ) and a phase delay of Cry1 (Fig. 4D ) and Ror α (Fig. 4H ). RF‐HF restored the phase of Clock (Fig. 4F ) and Cry1 (Fig. 4D ) and phase‐advanced all the other genes tested (Fig. 4 ). RF‐LF caused a phase advance in all clock genes and did not exhibit any phase delays compared with those in RF‐HF mice, except in Bmal1 and Cry1 mRNA (Fig. 4C, D ). Taken together, these results show that timed feeding is dominant and can rectify and/or advance the shifts induced by the HF diet.

Locomotor activity of mice fed the AL‐LF, AL‐HF, RF‐LF, and RF‐HF diet. A ) Representative double‐plotted actograms during LD and DD conditions. B ) Mean locomotor activity of mice fed the AL‐HF and RF‐HF diet during the last 10 d of LD conditions. Inset: total daily activity. C ) Mean locomotor activity of mice fed the RF‐LF and RF‐HF diet during the last 10 d of LD. Inset: total daily activity. Values are means ± se; n = 6/group. Shaded and solid bars designate the subjective day and night, respectively; cross‐hatched bar indicates food availability during RF. ∗ P < 0.05.

We next tested the effect of timed HF feeding on locomotor activity, a direct readout of the SCN clock. Activity of mice under AL feeding was initiated at the beginning of the dark phase and was mainly nocturnal ( Fig. 3 ). As expected, locomotor activity was changed in the timed groups (RF‐LF and RF‐HF); i.e ., before the time of food availability, mice displayed food anticipatory activity (Fig. 3A ). Total locomotor activity was higher under RF than under AL because of the biphasic activity occurring at the beginning of feeding time and at the beginning of the dark phase (Fig. 3 ). Of interest, the RF‐HF group was less active than the RF‐LF‐fed mice (Fig. 3B ). Under DD, mice exhibited free‐running behavior with a period of 23.8 ± 0.04 h in all groups (Fig. 3A ). Taken together, these results show that the timed HF diet leads to slight alterations in locomotor activity.

Leptin, a satiety‐inducing hormone secreted from adipose tissue, oscillated under all diets (data not shown) and exhibited higher daily levels in the AL groups compared with the RF groups (Fig. 2G ). Although fat mass was similar in both RF groups, leptin levels were 2‐fold higher in the RF‐HF group, indicating increased satiety compared with that for the RF‐LF group ( P <0.05, Tukey's HSD; Fig. 2G ). Corticosterone, a stress hormone, and ghrelin, a hunger‐inducing hormone, oscillated under all diets except for corticosterone in the RF‐HF group (data not shown). The daily levels of these hormones in the RF‐HF group were not significantly different from those in the AL groups (Fig. 2H, I ). However, the timed HF diet led to 53 and 25% lower ( P <0.05, Tukey's HSD) corticosterone and ghrelin levels, respectively, compared with those for the RF‐LF group, indicating reduced hunger and stress. Adiponectin oscillated only in the LF groups (data not shown) and did not show a significant difference in total daily levels among the RF‐HF, RF‐LF, and AL‐HF groups ( P <0.05, Tukey's HSD; Fig. 2J ). IL‐6 oscillated under all diets, but TNF‐α did not show robust oscillation except for RF‐LF mice ( P <0.05, Tukey's HSD; data not shown). The daily levels of TNF‐α, an inflammatory marker, were ∼10% up‐regulated in the AL‐HF group, whereas RF‐HF led to levels similar to those of the AL‐LF group (Fig. 2K ), indicating reduced inflammation. No change was observed in IL‐6 levels ( P <0.05, Tukey's HSD; Fig. 2L ). Taken together, these results show that as opposed to RF‐LF mice, the timed HF diet shows increased satiety and reduced hunger and stress.

Glucose and triglycerides exhibited a daily rhythm (data not shown) and their levels were not significantly different between the AL‐HF and RF‐HF groups ( P <0.05, Tukey's HSD; Fig. 2A, B ). The RF‐HF group exhibited 24 and 25% higher levels of glucose and triglycerides, respectively, than the RF‐LF group; however, these levels were within the normal range. Total cholesterol (Fig. 2C ) and HDL (Fig. 2D ) daily levels in the RF‐HF group were 21 and 32% lower, respectively ( P <0.05, Tukey's HSD), than those for the AL‐fed groups and similar to those for the RF‐LF group. Insulin exhibited circadian oscillation in all groups (data not shown). However, AL‐HF mice exhibited high insulin (Fig. 2E ) and HOMA‐IR (Fig. 2F ) levels, which are indicative of reduced insulin sensitivity. In contrast, the RF‐HF diet led to levels of insulin and HOMA‐IR ( P <0.05, Tukey's HSD) similar to those of the AL‐LF group. Taken together, these results show that timed HF diet leads to reduced cholesterol levels and prevents or delays considerably the development of insulin resistance as opposed to the AL‐HF diet.

All 4 groups (AL‐LF, AL‐HF, RF‐LF, and RF‐HF) gained weight throughout the experiment, with a final body weight greater in the HF groups than in the LF groups ( Fig. 1A ). Interestingly, body weight of the RF‐HF group was 12% lower than that of the AL‐LF group. Because the amount of calories per gram of food was higher for the HF diet, we calculated food consumption in kilocalories. As expected, AL‐HF mice consumed more calories than all the other groups. Interestingly, the RF‐HF group consumed the same amount of calories as the AL‐LF mice. In addition, food consumption per lean body mass (body weight 0.75 ) was 91% in grams and 106% in calories for the RF‐HF group, showing no caloric restriction (Fig. 1B ). As expected from their lower body weight, epididymal fat was lower in the RF groups than in the AL‐LF group (Fig. 1C ). Surprisingly, although body weight and epididymal fat mass were ∼20 and 48% lower, respectively, in the RF‐HF group than in the AL‐HF group, liver lipid content was not significantly different between these groups (Fig. 1D ). Taken together, these results show that RF‐HF diet leads to lower body weight and epididymal fat stores than the AL‐LF diet.

To examine the effect of timed HF feeding (RF‐HF) on circadian metabolism and obesity, mice were fed regular chow for 4 h every day at the same time for 18 wk and were compared with mice fed a timed LF diet (RF‐LF) and mice fed an HF diet AL (AL‐HF). Our baseline was another group fed an LF diet AL (AL‐LF). For all analyses, 6 time points throughout the circadian cycle were used to measure oscillation as well as average daily levels for a more accurate assessment of protein and/or mRNA levels.

DISCUSSION

Our results show that the timed HF diet leads to a unique metabolic phenotype of calorie intake equal to that of AL‐LF mice but with reduced body weight. The total activity of RF‐HF mice was higher than that of AL‐HF and lower than that of RF‐LF mice, correlating with their body weight. Although AL‐HF mice exhibited elevated cholesterol levels and fat depots compared with LF‐fed mice, timing of the HF diet reversed these effects. Nevertheless, liver lipid content was similar whether the HF diet was AL or timed. This finding suggests that the high lipid content in the liver was exported for storage in fat depots in AL‐HF mice, but was used in RF‐HF mice during the time mice were devoid of food, as indeed was reflected by the increased pACC and Pparα mRNA levels (see below).

Hormonal analyses reflect quite clearly the fact that RF‐HF mice exhibit a satiated state. Levels of serum ghrelin, a hunger‐inducing peptide (32,33) were not significantly different from those in AL‐HF mice but were lower compared with those in RF‐LF mice. In addition, the levels of leptin, which functions as a satiety signal whose levels are correlative with fat tissue mass (34), were high in AL‐HF mice and low in RF‐HF mice, indicating that the timed HF diet may prevent leptin resistance seen in the AL‐HF group. The timed HF diet also led to lower HOMA‐IR values compared with those in AL‐HF mice, representing higher insulin sensitivity. Metabolic alterations as a result of the RF regimen are associated with stress, as food is introduced for a short period of time, as indicated by the high corticosterone levels. On the other hand, studies have suggested that the HF diet selectively protects against the effects of chronic stress (35,36), as exhibited by the low corticosterone levels in HF‐fed mice. Thus, the low leptin levels together with no change in corticosterone and ghrelin levels in RF‐HF mice compared with AL‐HF mice indicate increased leptin sensitivity and a nonstressed, satiated status. The reduced locomotor activity in RF‐HF mice compared with that in RF‐LF mice may also indicate that the animals were more satiated and less stressed. Timed RF also led to reduced levels of TNF‐α compared with those in AL‐HF mice and similar to those of AL‐LF mice. Because TNF‐α serves as a marker of inflammation, our results indicate that timing can attenuate the detrimental impact of the HF diet and restore inflammatory levels to those of AL‐LF.

At the cellular level, under RF‐LF, mice were devoid of food for 20 h, leading to increased AMP levels and subsequent AMPK activation, which leads to reduced fatty acid synthesis and increased fatty acid oxidation in the liver, as was reported previously (37). Similarly to AMPK, SIRT1 activity is up‐regulated in response to changes in the energy status (38). Levels of pAMPK, the activated form of AMPK, pACC, and SIRT1 daily average levels did not change in the liver in RF‐HF mice compared with those in AL‐HF mice, indicating that restricting food availability to 4 h did not lead to reduced energy levels. Moreover, the timed HF diet led to pAMPK levels lower than those of RF‐LF mice and increased pACC levels compared with those in AL‐LF mice, indicating adequate energy levels but reduced fatty acid synthesis. Increased fatty acid oxidation was also reflected by the increased daily levels of Pparα mRNA under the timed HF diet. Thus, at the cellular levels, the timed HF diet led to the activation of catabolic pathways although energy levels were adequate. These findings support the reduced body weight of RF‐HF mice.

The clock proteins CLOCK, BMAL1, PER1, PER2, CRY1, and CRY2 have been shown to be important for regulated metabolism. Mutation or knockout of clock genes has led to metabolic disturbances (6,8,13,39,40). Moreover, we and others have shown that an HF diet leads to changes in behavioral rhythmicity and correlates with disrupted circadian gene expression within hypothalamus, liver, and adipose tissue (21-26). Indeed, our data show that the AL‐HF diet disrupted the circadian expression of some clock genes (Fig. 4). However, the RF‐HF diet restored the phase of some clock genes and phase‐advanced the others. Similar effects were found with metabolic genes and proteins. Thus, timed feeding is dominant (18-20) and can rectify and/or advance the shifts induced by an HF diet. It was recently reported that mice fed an HF diet during the light phase gain significantly more weight than mice fed only during the dark period (27). Our results emphasize the importance of timed feeding of 4 h vs. food availability of 12 h. Indeed, we have recently shown the benefits in timed vs. ad libitum feeding (17).

In summary, our findings show that the timed HF diet leads to increased insulin sensitivity and fat oxidation and decreased body weight, fat profile, and inflammation contrary to HF‐diet‐fed mice but comparable to LF‐diet‐fed mice. Because an HF diet is difficult to abstain from, the timing of meals can be suggested for individuals seeking weight loss and better reset metabolism.