Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus

Abstract

In mammals, circadian oscillators exist not only in the suprachiasmatic nucleus, which harbors the central pacemaker, but also in most peripheral tissues. It is believed that the SCN clock entrains the phase of peripheral clocks via chemical cues, such as rhythmically secreted hormones. Here we show that temporal feeding restriction under light-dark or dark-dark conditions can change the phase of circadian gene expression in peripheral cell types by up to 12 h while leaving the phase of cyclic gene expression in the SCN unaffected. Hence, changes in metabolism can lead to an uncoupling of peripheral oscillators from the central pacemaker. Sudden large changes in feeding time, similar to abrupt changes in the photoperiod, reset the phase of rhythmic gene expression gradually and are thus likely to act through a clock-dependent mechanism. Food-induced phase resetting proceeds faster in liver than in kidney, heart, or pancreas, but after 1 wk of daytime feeding, the phases of circadian gene expression are similar in all examined peripheral tissues.

Figure 1

Daytime feeding changes the phase of circadian gene expression in the liver but not in the suprachiasmatic nucleus (SCN). Mice, kept under a light-dark regimen (lights on 6 a.m., lights off 6 p.m.), were fed for eight consecutive days exclusively during the light phase (6 a.m. to 6 p.m.) or during the dark phase (6 p.m. to 6 a.m.). During the ninth day, animals were killed at 4-h intervals to prepare whole-cell RNA from liver and tissue sections from the brain. (A) Circadian accumulation of clock and clock-controlled genes in the liver. The levels of transcripts from the genes indicated to the left of the panels were determined by ribonuclease protection assays (Cyp2a5 stands for coumarin hydroxylase). Tbp mRNA or β-actin mRNA were included as standards for transcripts whose expression is constant throughout the day (data not shown). (B) The mRNA signals obtained in the ribonuclease protection assays shown in panel A were measured by scanning the autoradiography and normalized to the signals generated by β-actin mRNA (for Cyp2a5) or Tbp mRNA (for all other transcripts). (C) Serial coronal brain sections taken above the optical chiasma were hybridized in situ to ³⁵S-labeled Per1 and Per2 antisense and sense RNA strands. Note that no detectable hybridization signals were obtained with the sense probe. (D) Circadian accumulation of Per1 and Per2 mRNA in the SCN of mice after 8 d of restricted feeding. Coronal sections from brains harvested at 4-h intervals around the clock were hybridized in situ to Per1 and Per2 antisense RNA probes as described in panel C. Note that the phases of rhythmic Per1 and Per2 expression are not affected by restricted feeding.

Figure 2

ADDER (amplification of double-stranded cDNA end restriction fragments) differential display of liver cDNAs from mice fed exclusively during the day or during the night. Double-stranded cDNA fragments encompassing mRNA sequences located between the poly A addition sites and the most proximal Mbo1 restriction sites were synthesized using total liver RNA as a template. The RNA was collected at 4-h intervals from mice fed either during the day or during the night. The left panel shows a subpopulation of cDNAs containing the 3′ sequences of coumarin 7 hydroxylase (Cyp2a5) transcripts and ∼60 other 3′-terminal mRNA sequences amplified and displayed on a 5% urea-polyacrylamide gel (see Materials and Methods). X is a yet unidentified circadian fragment. The right panel shows a subpopulation of cDNA amplified using a second set of primers (see Materials and Methods). Y is a yet unidentified circadian fragment. The positions of molecular size markers are indicated in the middle of the figure.

Figure 3

Circadian gene expression in the liver and the suprachiasmatic nucleus (SCN) of food-entrained mice kept in constant darkness. Mice, kept in constant darkness (DD) were fed for six consecutive days exclusively during the subjective day (6 a.m. to 6 p.m.) or during the subjective night (6 p.m. to 6 a.m.). During the seventh day, animals were killed at 4-h intervals to prepare whole-cell RNA from liver and tissue sections from the brain. (A) Circadian Per1 and Per2 mRNA accumulation in the liver. Transcript levels were determined as described in Figure 1. (B) The signals obtained in the ribonuclease protection assays shown in panel A were quantified and normalized to Tbp mRNA levels (not shown in panel A). (C) The accumulation of Per1 and Per2 mRNA in the SCN of food entrained animals was revealed by in situ hybridization as described in Figure 1.

Figure 4

Circadian gene expression changes gradually during restricted feeding. (A) Mice kept under a LD regimen (lights on at 6 a.m., lights off at 6 p.m.) were fed exclusively during the day for 1 d, 3 d, or 1 wk. The animals were then sacrificed at 2 and 10 p.m. to prepare whole-cell liver RNA. Transcript levels for Cry1 and Rev-erbα were determined as described in Figure 1. (B) Mice were fed exclusively during the day for seven consecutive days and, after a starvation of 24 h, switched to nighttime feeding by offering food during the night of the eighth day (from 6 p.m. to 6 a.m.). Animals were sacrificed at 4-h intervals (starting at 6 p.m. of the eighth day) for the analysis of mRNAs. Note that Per2 mRNA, Rev-erbα mRNA, and Cry1 mRNA are still at levels intermediate between those observed for mice fed during the night or during the day.

Figure 5

Circadian gene expression in liver after food-entrainment. (A) Mice kept under a LD regimen (lights on at 6 a.m., lights off at 6 p.m.) were fed exclusively during the day for seven consecutive days. On the morning of the eighth day, food was not given back, and animals were sacrificed at 4-h intervals during the following 36 h. Thus, after daytime feeding the mice were starved between 12 and 48 h before they were killed for the analysis of the various mRNAs (depicted to the left of the panels). (B) mRNA levels obtained for starved mice (panel A) were quantified and normalized to Tbp mRNA. The times at which zenith mRNA levels were reached in daytime- and nighttime-fed mice are indicated by black and white arrows, respectively (see Fig. 1). (C) Per1 and Per2 mRNA accumulation in the suprachiasmatic nucleus (SCN) of starved animals (see panel A) has been determined by in situ hybridization to coronal brain sections as described in Figure 1. Note that daytime feeding followed by starvation has no effect on circadian Per1 and Per2 mRNA accumulation in the SCN.

Figure 6

Restricted feeding resets the phase in various peripheral tissues. Mice kept under a light-dark regimen (lights on 6 a.m., lights off 6 p.m.) were fed for three or six consecutive days exclusively during the light phase (6 a.m. to 6 p.m.) or for six days exclusively during the dark phase (6 p.m. to 6 a.m.). During the fourth or seventh day, respectively, animals were sacrificed at 4-h intervals to prepare whole-cell RNA from liver, kidney, heart, and pancreas. Dbp mRNA levels were determined by ribonuclease protection assays in the organs and at the daytimes indicated on top of the panels. β-actin mRNA was included as a transcript whose accumulation does not oscillate during the day. (A) Temporal Dbp expression in mice fed during 6 d exclusively during the night. (B) Temporal Dbp expression in mice fed during 6 d exclusively during the day. (C) Temporal Dbp expression in mice fed during 3 d exclusively during the day.

Figure 7

Daytime feeding causes body temperature depressions during the night. Temperature probes were implanted in the abdominal cavities of mice and body temperature rhythms were recorded by telemetry in animals fed either ad libitum or exclusively during the night or during the day. The feeding periods are indicated by solid bars on top of the panels. The dark phases are depicted in blue. (A) Body temperature recordings from two individuals fed during 5 d exclusively during the day. Note the dramatic temperature depressions during the dark phases of daytime feeding. (B) Recordings from two individuals fed during 7 d exclusively during the night. In some animals (e.g., the one shown in the bottom panel), the minimal temperature values observed during the light phase became slightly lower after several days of nighttime feeding.

Figure 8

Hypothetical model on the entrainment of peripheral oscillators. The central circadian pacemaker in the suprachiasmatic nucleus (SCN) is entrained by solar cycles via the retino-hypothalamic tract (RHT). (A) When food is available throughout the day or during the normal activity phase (dark phase in nocturnal animals), the SCN synchronizes peripheral clocks via cycles in the secretion of blood-borne factors (e.g., hormones) or temperature rhythms. Both of these time cues may ultimately be controlled by the feeding behavior governed by the SCN. (B) When food is only available during the phase at which the animals are normally inactive (light phase in nocturnal animals), signals triggered by food processing and/or the lack of food processing act as dominant Zeitgebers on the oscillators of peripheral tissues.