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. 2012 Aug 15;303(4):G461-73.
doi: 10.1152/ajpgi.00369.2011. Epub 2012 Jun 21.

Circadian Rhythms of Gastrointestinal Function Are Regulated by Both Central and Peripheral Oscillators

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Free PMC article

Circadian Rhythms of Gastrointestinal Function Are Regulated by Both Central and Peripheral Oscillators

Jaclyn N Malloy et al. Am J Physiol Gastrointest Liver Physiol. .
Free PMC article

Abstract

Circadian clocks are responsible for daily rhythms in a wide array of processes, including gastrointestinal (GI) function. These are vital for normal digestive rhythms and overall health. Previous studies demonstrated circadian clocks within the cells of GI tissue. The present study examines the roles played by the suprachiasmatic nuclei (SCN), master circadian pacemaker for overt circadian rhythms, and the sympathetic nervous system in regulation of circadian GI rhythms in the mouse Mus musculus. Surgical ablation of the SCN abolishes circadian locomotor, feeding, and stool output rhythms when animals are presented with food ad libitum, while restricted feeding reestablishes these rhythms temporarily. In intact mice, chemical sympathectomy with 6-hydroxydopamine has no effect on feeding and locomotor rhythmicity in light-dark cycles or constant darkness but attenuates stool weight and stool number rhythms. Again, however, restricted feeding reestablishes rhythms in locomotor activity, feeding, and stool output rhythms. Ex vivo, intestinal tissue from PER2::LUC transgenic mice expresses circadian rhythms of luciferase bioluminescence. Chemical sympathectomy has little effect on these rhythms, but timed administration of the β-adrenergic agonist isoproterenol causes a phase-dependent shift in PERIOD2 expression rhythms. Collectively, the data suggest that the SCN are required to maintain feeding, locomotor, and stool output rhythms during ad libitum conditions, acting at least in part through daily activation of sympathetic activity. Even so, this input is not necessary for entrainment to timed feeding, which may be the province of oscillators within the intestines themselves or other components of the GI system.

Figures

Fig. 1.
Fig. 1.
Light cycles and feeding regimens of operated animals. A: intact bilateral suprachiasmatic nuclei (SCN) immunostained for VIP in a sham-operated animal. B: section from a mouse in which the SCN was electrolytically lesioned (SCNX). VIP staining was not visible in negative controls (data not shown). C and D: actograms showing circadian locomotor activity. C: rhythmic circadian locomotor activity of a sham-operated animal injected with ascorbate. D: arrhythmic locomotor behavior of a SCNX animal injected with ascorbate. Arrows indicate beginning of a new light cycle: black arrows show light-dark (LD) cycles, and gray arrows show constant darkness (DD). Boxes indicate 4 h of midday food availability during restricted feeding (RF).
Fig. 2.
Fig. 2.
Food consumption and stool output are rhythmic in sham (n = 8) and arrhythmic in SCNX (n = 10) animals during ad libitum food availability. A–C: food consumption, stool weight, and stool number in SCNX and sham animals during 3 days in LD. ZT, zeitgeber time. D–F: food consumption, stool weight, and stool number in SCNX and sham animals during 3 days in DD. CT, circadian time. For determination of circadian rhythmicity of a data set, peak values were compared with the corresponding following and/or preceding trough by 1-way ANOVA. *Significance in sham animals (P < 0.05). No significance was reported in SCNX animals. Values are means ± SE.
Fig. 3.
Fig. 3.
Colonic motility in SCNX and sham animals directly entrains to food availability. Food was available for 4 h daily (from 12 PM to 4 PM) during ZT0–ZT4 (vertical gray bars in B and C). Bars along the x-axis in B and C represent the previous LD cycle. A: food consumption during each day of RF in SCNX and sham mice. B: stool weight during RF. C: stool number during RF. *P < 0.05. No significant difference was shown by 2-way ANOVA between groups in A. Values are means ± SE.
Fig. 4.
Fig. 4.
SCN lesion yields arrhythmic food consumption and stool output during ad libitum food availability in 6-hydroxydopamine (6-OHDA)-treated (n = 6) and vehicle-treated (n = 4) mice. A–C: food consumption, stool weight, and stool number in SCNX animals during LD. D–F: food consumption, stool weight, and stool number in SCNX animals during DD. Values are means ± SE.
Fig. 5.
Fig. 5.
Differences in stool output rhythms between 6-OHDA-treated (n = 3) and vehicle-treated (control, n = 5) sham animals during ad libitum conditions. AC: food consumption, stool weight, and stool number in 6-OHDA- and vehicle-treated sham animals in LD. D–F: food consumption, stool weight, and stool number in 6-OHDA- and vehicle-treated sham animals during DD. *P < 0.05 for sham control. *P < 0.05 for sham 6-OHDA. Values are means ± SE.
Fig. 6.
Fig. 6.
Colonic motility in all treated animals directly entrains to food availability. Food was available for 4 h daily (from 12 PM to 4 PM) during ZT0–ZT4 (vertical gray bars in B–E). A: food consumption of each surgery/treatment group. B and C: stool weight and stool number of SCNX animals during RF. D and E: stool weight and stool number of sham animals during RF. *P < 0.05 for sham control. *P < 0.05 for sham 6-OHDA. No significant difference (by 2-way ANOVA) in food consumption was shown between groups in A. Values are means ± SE.
Fig. 7.
Fig. 7.
SCN lesion and 6-OHDA treatment do not decrease the number of tyrosine hydroxylase (TH)-expressing neurons within the arcuate nucleus, but 6-OHDA treatment does decrease TH immunoreactivity in the colon. A–D: representative histological sections showing TH immunohistochemical staining of the arcuate nucleus in the brain of an ascorbate-treated sham animal, a 6-OHDA-treated sham animal, an ascorbate-treated SCNX animal, and a 6-OHDA-treated SCNX animal. E and F: representative distal colonic sections showing immunofluorescent detection of TH-expressing neurons in ascorbate-treated (E) and 6-OHDA-treated (F) animals. Scale bars, 100 μm. G: representative Western blot showing immunoreactive TH. Left to right, 6-OHDA-treated tissues (lanes 1 and 2) and ascorbate-treated tissues (lanes 3 and 4). H: average composite of relative density of experimental and control immunoreactive TH bands of 4 independent Western blots.
Fig. 8.
Fig. 8.
6-OHDA has little effect and isoproterenol causes a phase shift in PER2 expression ex vivo. A: period and damping factor in tissues from vehicle-treated (n = 4) and 6-OHDA-treated (n = 4) animals. Values are means ± SE. *P < 0.05 (Student's t-test). B: representative traces of baseline-subtracted luciferase bioluminescence [in counts per second (cps)] from tissues of vehicle-treated (control) and 6-OHDA-treated animals. C: representative traces of baseline-subtracted luciferase bioluminescence from tissues treated with a pulse of isoproterenol or vehicle solutions. Phase resetting occurs after treatment (gray arrow) in isoproterenol-treated, but not control, tissues. D: phase-response curve generated using PER2 expression data from isoproterenol- and vehicle-treated tissues. *P < 0.005; +P < 0.001 (by 2-way ANOVA). Phase shifting is dependent on time of isoproterenol treatment in relation to peak PER2 expression.

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