Gut microbe-derived trimethylamine shapes circadian rhythms through the host receptor TAAR5

We recently demonstrated that drugs blocking gut microbial TMA production protect against obesity via rewiring circadian rhythms in the gut microbiome, liver, white adipose tissue (WAT), and skeletal muscle (Schugar et al., 2022). Furthermore, we also showed that blocking bacterial TMA production elicited unexpected alterations in olfactory perception of diverse odorant stimuli (Massey et al., 2023). Therefore, we have followed up here to further interrogate circadian rhythms in the gut microbiome, liver, WAT, skeletal muscle, and olfactory bulb in mice lacking the only known host G-protein-coupled receptor (GPCR) that senses TMA known as TAAR5 (Wallrabenstein et al., 2013; Li et al., 2013). In agreement with our previous report showing that Taar5 mRNA is expressed in a circadian manner in skeletal muscle (Schugar et al., 2022), LacZ reporter expression oscillates with peak expression in the dark cycle in skeletal muscle (Figure 1A). However, unlike the striking impact that choline TMA lyase inhibitors have on the core circadian clock machinery in skeletal muscle (Schugar et al., 2022), mice lacking the TMA receptor (Taar5-/-) also have largely unaltered circadian gene expression in skeletal muscle, with only nuclear receptor subfamily 1 group D member 1 (Nr1d1, Rev-Erbα) showing a modest yet significant delay in acrophase (Figure 1A; Supplementary file 1). Instead, Taar5-/- mice have alterations in the expression of key circadian genes including basic helix-loop-helix ARNT like 1 (Bmal1), clock circadian regulator (Clock), Nr1d1, cryptochrome 1 (Cry1), and period 2 (Per2) in the olfactory bulb (Figure 1B; Supplementary file 1). Taar5-/- mice exhibited increased mesor for Bmal1, Clock, and Nr1d1 compared to Taar5+/+ controls in the olfactory bulb (Figure 1B; Supplementary file 1). Whereas, Taar5-/- mice exhibited advanced acrophase in Cry1 and Per2 compared to Taar5+/+ controls in the olfactory bulb (Figure 1B; Supplementary file 1). There is also some reorganization of circadian gene expression in the liver (Figure 1C) and gonadal WAT (Figure 1D; Supplementary file 1), albeit modest. In the liver, Taar5-/- mice had normal circadian gene expression when compared to Taar5+/+ controls (Figure 1C; Supplementary file 1). In gonadal WAT, the amplitude of Bmal1 was increased and acrophase of Bmal1 was delayed in Taar5-/- mice, whereas only the acrophase of Per2 was advanced in Taar5-/- mice compared to Taar5+/+ controls (Figure 1D; Supplementary file 1). Given the key role that the TMAO pathway plays in suppressing the beiging of WAT (Schugar et al., 2017), we also examined the expression of PR/SET domain 16 (Prdm16) and uncoupling protein 1 (Ucp1). Taar5-/- mice had increased mesor and advanced acrophase of Prdm16, yet no difference was detected in Ucp1, compared to Taar5+/+ controls (Figure 1D; Supplementary file 1). Taar5-/- mice have unaltered oscillations in body weight (Figure 1—figure supplement 1; Supplementary file 1).

The host trimethylamine receptor TAAR5 shapes tissue-specific circadian oscillations.

Male chow-fed wild-type (Taar5+/+) or mice lacking the TMA receptor (Taar5-/-) were necropsied at 4-hr intervals to collect tissues including skeletal muscle (A), olfactory bulb (B), liver (C), or gonadal white adipose tissue (D). The relative gene expression for circadian (Bmal1, Clock, Nr1d1, Cry1, and Per2) and metabolism (Prdm16 and Ucp1) related genes was quantified by qPCR using the ΔΔ-CT method. Data shown represent the means ± SEM for n = 3–6 individual mice per group. Group differences were determined using cosinor analyses, and p-values are provided where there were statistically significant differences between Taar5+/+ and Taar5-/- mice. The complete cosinor statistical analysis for circadian data can be found in Supplementary file 1. *Significant differences between Taar5+/+ and Taar5-/- mice by Student’s t-tests within each ZT time point (p < 0.05).

We next examined circadian oscillations in circulating metabolite, hormone, and cytokine levels in Taar5+/+ and Taar5-/- mice (Figure 1—figure supplement 2; Supplementary file 1). When we measured substrates for gut microbial TMA production, we found that Taar5-/- mice had normal oscillations in choline and l-carnitine (Figure 1—figure supplement 2A; Supplementary file 1). However, there was a modest yet significant advance in the acrophase of plasma γ-butyrobetaine in Taar5-/- mice (Figure 1—figure supplement 2A; Supplementary file 1). Taar5-/- mice also had slightly increased levels of TMA at ZT22, when compared to Taar5+/+ controls (Figure 1—figure supplement 2A; Supplementary file 1). However, TMA and TMAO levels were not significantly altered in Taar5-/- mice (Figure 1—figure supplement 2A; Supplementary file 1). Interestingly, Taar5-/- mice had altered rhythmic levels in some but not all host metabolic hormones. Although the circadian oscillations of plasma insulin and C-peptide were not significantly altered, Taar5-/- mice had a delay in acrophase of plasma glucagon, yet advanced acrophase of glucagon-like peptide 1 (GLP-1), compared to Taar5+/+ controls (Figure 1—figure supplement 2B; Supplementary file 1). Taar5-/- mice also exhibited increased mesor for plasma leptin levels compared to wild-type controls (Figure 1—figure supplement 2B; Supplementary file 1). Taar5-/- mice also had modest differences in circulating cytokines, including a decrease in the mesor of monocyte chemoattractant 1 (MCP-1) and tumor necrosis factor α (TNFα) (Figure 1—figure supplement 2B; Supplementary file 1). Collectively, these data demonstrate that Taar5-/- mice have altered circadian-related gene expression that is most apparent in the olfactory bulb (Figure 1; Supplementary file 1), and abnormal circadian oscillations in some but not all circulating hormones and cytokines (Figure 1—figure supplement 2; Supplementary file 1).

We next set out to comprehensively analyze the circadian rhythms in behavioral phenotypes in mice lacking the TMA receptor TAAR5. In this line of investigation, we took a very broad approach to examine impacts of Taar5 deficiency on metabolic, cognitive, motor, anxiolytic, social, olfactory, and innate behaviors. The main rationale behind this in-depth investigation was to allow for comparison to our recent work showing that pharmacologic blockade of the production of the TAAR5 ligand TMA (using choline TMA lyase inhibitors) produced clear metabolic, innate, and olfactory-related social behavioral phenotypes, but did not dramatically impact aspects of cognition, motor, or anxiolytic behaviors (Schugar et al., 2022; Massey et al., 2023). Also, it is important to note other groups have recently shown that TAAR5 activation with non-TMA ligands or genetic deletion of Taar5 results in clear olfactory (Li et al., 2013; Liberles, 2015; Freyberg and Saavedra, 2020; Espinoza et al., 2020), anxiolytic (Espinoza et al., 2020), cognitive (Maggi et al., 2022), and sensorimotor (Aleksandrov et al., 2019; Kalinina et al., 2021) behavioral abnormalities. Here, it was our main goal to identify whether any behavioral phenotypes that were consistently seen in mice lacking bacterial TMA production (Schugar et al., 2022; Massey et al., 2023) or host TMA sensing by TAAR5 (studied here) are time-of-day dependent indicating circadian inputs. Given the consistent alterations in innate and olfactory-related phenotypes seen in both mice lacking bacterial TMA production (Schugar et al., 2022; Massey et al., 2023) and mice lacking host TMA sensing by TAAR5 (Wallrabenstein et al., 2013; Li et al., 2013; Liberles, 2015; Freyberg and Saavedra, 2020; Espinoza et al., 2020; Maggi et al., 2022; Aleksandrov et al., 2019; Kalinina et al., 2021), we next followed up to perform select innate and olfactory-related behavioral tests in mice at defined circadian time points (Figure 2).

Mice lacking the host TMA receptor TAAR5 have altered olfactory and repetitive behaviors only at specific circadian time points.

Male or female wild-type mice (Taar5+/+) or mice lacking the TMA receptor (Taar5-/-) were subjected to the olfactory cookie test (A) or the marble burying test (B). To examine circadian alterations in behavior, these tests were done in either the dark-light phase transition (ZT23–ZT1), mid light cycle (ZT5–ZT7), or early dark cycle (ZT13–ZT15). Data represent the mean ± SEM from n = 10–15 per group when male and female are separated (n = 25–27 when both sexes are combined), and statistically significant difference between Taar5+/+ and Taar5-/- mice are denoted by *p < 0.05 and **p < 0.01.

To study the circadian presentation of phenotype, we carefully controlled the time window of testing for either the olfactory cookie test or marble burying test, both of which have been shown to be altered in mice lacking bacterial TMA synthesis (Massey et al., 2023; Romano et al., 2017). When the olfactory cookie test was performed during the mid-light cycle (ZT5–ZT7), the latency to find the buried cookie was significantly increased in male Taar5-/- mice compared to Taar5+/+ controls (Figure 2A). However, when the same mice performed the olfactory cookie test at the dark-to-light phase transition (ZT23–ZT1), or at the light-to-dark phase transition (ZT13–ZT15), there were no significant differences between Taar5+/+ and Taar5-/- mice (Figure 2A). When subjected to the marble burying test, only female Taar5-/- mice buried significantly more marbles than female wild-type controls only at ZT5–ZT7, but this was not apparent at other ZT time points (Figure 2B). Collectively, these data demonstrate that Taar5-/- mice exhibit highly gender-specific alterations in innate and olfactory behaviors, and these behavior phenotypes are only apparent at certain periods within the light cycle (Figure 2 and S2).

Although Taar5-/- mice showed time-dependent alterations in the olfactory cookie test, olfactory discrimination toward other diverse single stimuli such as banana, corn oil, almond, water, or social cues was not significantly altered (Figure 2—figure supplement 1). When we subjected Taar5-/- mice to a battery of social behavioral tests, there were test-specific alterations that occurred in a sexually dimorphic manner. All mouse groups (Taar5+/+, Taar5-/-, male and female) displayed no initial chamber bias in the initial trial of the three-chamber test (Figure 2—figure supplement 2A). In the three-chamber preference test, only male Taar5-/- mice showed no preference between an inanimate object and a social stimuli interaction (Figure 2—figure supplement 2B). When subjected to the three-chamber social novelty test box, both male and female Taar5-/- mice showed no preference between the novel and familiar stimuli (Figure 2—figure supplement 2C). In the social interaction with a juvenile mouse test, Taar5-/- females showed no significant difference in interaction time between the initial interaction trial and the recognition trial 4 days later (Figure 2—figure supplement 2D). In addition to alterations in social interactions, Taar5-/- mice also showed sexually dimorphic alterations in several other innate behavioral tests (Figure 2—figure supplement 3). Female Taar5-/- mice exhibited a significantly higher startle response at 90, 100, 110, and 120 decibels (Figure 2—figure supplement 3A), and significantly weaker forelimb grip strength (Figure 2—figure supplement 3B) compared to Taar5+/+ controls. When both sexes are combined, there is a significant increase in the latency to withdraw during the hotplate sensitivity test in Taar5-/- mice compared to controls (Figure 2—figure supplement 3C). Furthermore, both male and female Taar5-/- mice have slightly increased latency to fall during the rotarod test (Figure 2—figure supplement 3D). Also, female but not male Taar5-/- mice exhibit reduced nest building compared to Taar5+/+ mice (Figure 2—figure supplement 3E).

We next comprehensively examined cognitive, depression, and anxiety-like behaviors in Taar5+/+ and Taar5-/- mice (Figure 2—figure supplements 4 and 5). Both male and female Taar5-/- mice performed similarly to Taar5+/+ controls in the open field, elevated plus maze, and Y-maze tests (Figure 2—figure supplement 4B–D). However, female, but not male, Taar5-/- mice showed significantly reduced freezing compared to Taar5+/+ controls in the cued fear conditioning test (Figure 2—figure supplement 4A). When subjected to the Morris water maze, there were only minor alterations found in Taar5-/- mice. All mice showed similar latency to the platform. Male Taar5-/- mice showed increased distance traveled compared to wild-type controls, yet females were more similar to Taar5+/+ controls (Figure 2—figure supplement 5). However, female Taar5-/- mice showed increased velocity compared to Taar5+/+ mice during the last 3 days of testing (Figure 2—figure supplement 5). Collectively, the impact of Taar5 deficiency on cognitive, depression, and anxiety-like behaviors was very modest.

Given the TMAO pathway has been linked to the beiging of WAT and energy expenditure (Schugar et al., 2017), we next examined circadian rhythms in energy metabolism during a cold challenge and gene expression in thermogenic brown adipose tissue (BAT) in Taar5-/- mice (Figure 2—figure supplement 6). Although male Taar5-/- mice showed unaltered oxygen consumption at thermoneutrality (30°C), room temperature (22°C), and during cold (4°C) exposure, female Taar5-/- mice had significantly elevated oxygen consumption that appeared most significant during the light cycle periods (Figure 2—figure supplement 6A). To follow up, we collected BAT from Taar5+/+ and Taar5-/- mice at ZT2 (early light cycle) and ZT14 (early dark cycle) to examine potential alterations in circadian gene expression. Both male and female Taar5-/- mice showed marked upregulation of Bmal1, yet other than increased Per1 expression at ZT14, all other circadian genes were largely unaltered in Taar5-/- mice (Figure 2—figure supplement 6B). Taken together, all behavioral data presented here show that mice lacking Taar5 have select sexually dimorphic alterations in olfactory, innate, social, and metabolic phenotypes.