Circadian Regulation of Peripheral Serotonin and Platelets in Mice

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2024-03-07

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Karaganis, Stephen
Zimmerman, Joey

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Abstract

Most organisms are known to possess biological clocks, which control and coordinate numerous physiological processes over each 24-hour day. Circadian oscillators play a role in generating biological rhythms and coordinating numerous processes with environmental stimuli, such as timing of a meal or exposure to light. The indolamine molecule, serotonin, is an important peripheral hormone produced by the intestinal mucosa of mammals, but its regulation as an output of the circadian clock is not well understood. Recent studies in our lab have investigated circadian rhythmicity of serotonin and its entrainment to light stimuli or food availability in various tissue compartments in mice, including blood serum, stools, and the intestinal wall. Because most serotonin is released into the blood and taken up by platelets, we investigated regulation of circulating platelet levels as well. In these experiments, mice were fed ad libitum (AL) or placed on a gradual daytime restricted feeding regimen (DRF) while maintained in a 12:12 light-dark cycle (LD) or constant darkness (DD). We assessed serotonin levels in duodenum, colon, and stool and demonstrated a high-amplitude circadian rhythm of serotonin in stool samples that persisted in constant conditions and entrained to both light and food availability, with a peak occurring close to the day-night transition under LD conditions. In contrast to some published findings, no circadian rhythm of serotonin was detected in blood serum. Serotonin levels from duodenum and colon also exhibited food-entrainable circadian rhythms, peaking in the early morning under LDRF. mRNA levels of tph 1, the rate-limiting enzyme for non-neuronal serotonin biosynthesis, was also rhythmic in the duodenum, and entrained to food availability, with a peak occurring approximately 16 hours prior to the peak in serotonin. This delay may reflect the kinetics of protein synthesis and turnover, as well as rising levels of serotonin transporter (SERT) measured in the late evening under RF conditions. Interestingly, a circadian rhythm in total platelet number was strongly entrained to cycles of food availability, but not to light.

Description

Circadian rhythms are 24-hour cycles of biological processes that persist under constant conditions. These rhythms are entrainable to external cues (known as zeitgebers), such as light or food availability, but persist in the absence of these cues. Nearly all organisms, including humans and other mammals, have circadian rhythms (1), which are outputs of oscillators comprised of transcriptional feedback loops (1,2). Central tissues are known to be strongly coupled with the light entrainable oscillator (LEO), located within the suprachiasmatic nucleus (SCN). This structure serves as a circadian pacemaker in mammals and other vertebrate species. On the other hand, peripheral tissues have generally been shown to be more strongly influenced by entrainment to food availability than to light cycles, thus more tightly coupled to the food entrainable oscillator (FEO) than to the LEO (3). However, this has not been directly demonstrated for all outputs of circadian oscillators, including peripheral serotonin. Serotonin, or 5-HT (5-hydroxytryptamine), derived from the essential amino acid tryptophan, is reported to be an output of the circadian clock in some species and tissues (4). Serotonin has been shown to be involved in a number of different physiological processes (4). For example, gut-derived serotonin can regulate stimulation of propulsive and segmentation motility patterns, epithelial secretion, vasodilation, immune function, and possibly skeletal growth (5,6). Most of the body's serotonin is transported into the blood before being taken up by platelets, and rhythms of serotonin abundance have also been measured in the plasma and serum of blood in various species including rodents (4). However, the processes by which peripheral serotonin is regulated by the circadian clock are not completely understood. The study of peripheral serotonin may give more valuable insight into the role of circadian clocks in regulating gastrointestinal, cardiovascular, and other functions. This research study aims to: 1. Determine how serotonin levels in the colon and duodenum of mice are regulated by environmental cycles of light or food availability. 2. Assess circadian control of circulating platelet levels under similar conditions. In these experiments, mice were fed ad libitum (AL) or placed on a gradual daytime restricted feeding regimen (DRF) while maintained in a 12:12 light-dark cycle (LD) or constant darkness (DD). During the sampling period, eight mice were sacrificed every four hours, beginning two hours after the start of the photophase, at ZT2. Whole trunk blood was be collected and diluted 1:100 with Rees and Ecker blood diluting fluid (Ricca Chemical, VWR Radnor, PA), then loaded into a hemocytometer for manual counting. Additional tissue samples (whole blood, liver, duodenum, jejunum, ileum, and colon) were taken from each animal, flash-frozen, and maintained at -80C. In a separate study, stool samples were collected every four hours using a repeated-measures design under similar conditions. Stool and gut samples were transported to TTUHSC for analysis using LC/MS/MS. mRNA levels of tph1 and SERT were quantified using qPCR. Cosinor statistical analysis was performed using CircWave software. References: 1. Bell-Pedersen D, Cassone VM, Earnest DJ, Golden SS, Hardin PE, Thomas TL, et al. Circadian rhythms from multiple oscillators: Lessons from diverse organisms. Nature Reviews Genetics. 2005Jun10;6(7):544-56. 2. Takahashi JS. Transcriptional architecture of the mammalian circadian clock. Nat Rev Genet. 2017 Mar;18(3):164-179. 3. Damiola F, Le Minh N, Preitner N, Kornmann Benoı̂t, Fleury-Olela F, Schibler U. Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus. Genes & Development. 2000;14(23):2950-61. 4. Valdés-Fuentes. "Effect of Daytime-Restricted Feeding in the Daily Variations of Liver Metabolism and Blood Transport of Serotonin in Rat." Physiological Reports, vol. 3, no. 5, 2015. 5. Mawe, Gary M., and Jill M. Hoffman. "Serotonin Signaling in the Gut-Functions, Dysfunctions and Therapeutic Targets." Nature Reviews Gastroenterology & Hepatology, vol. 10, no. 8, 2013, pp. 473-486. 6. Ebert-Zavos E, Horvat-Gordon M, Taylor A, Bartell PA. Biological clocks in the duodenum and the diurnal regulation of duodenal and plasma serotonin. PLoS One. 2013 May 30;8(5):e58477.

Keywords

2024 Faculty and Student Research Poster Session and Research Fair, West Texas A&M University, College of Agriculture and Natural Science, Poster, Circadian rhythms, Serotonin, Mice

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https://hdl.handle.net/11310/6382

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2024 Faculty and Student Research Poster Session and Research Fair