Abstract

Elevated levels of the gut microbe-derived metabolite trimethylamine N-oxide (TMAO) are associated with cardiometabolic disease risk. However, the mechanism(s) linking TMAO production to human disease are incompletely understood. Initiation of the metaorganismal TMAO pathway begins when dietary choline and related metabolites are converted to trimethylamine (TMA) by gut bacteria. Gut microbe-derived TMA can then be further oxidized by host flavin-containing monooxygenases to generate TMAO. Previously, we showed that drugs lowering both TMA and TMAO protect mice against obesity via rewiring of host circadian rhythms (Schugar et al., 2022). Although most mechanistic studies in the literature have focused on the metabolic end product TMAO, here we have instead tested whether the primary metabolite TMA alters host metabolic homeostasis and circadian rhythms via trace amine-associated receptor 5 (TAAR5). Remarkably, mice lacking the host TMA receptor (Taar5−/−) have altered circadian rhythms in gene expression, metabolic hormones, gut microbiome composition, and diverse behaviors. Also, mice genetically lacking bacterial TMA production or host TMA oxidation have altered circadian rhythms. These results provide new insights into diet–microbe–host interactions relevant to cardiometabolic disease and implicate gut bacterial production of TMA and the host receptor that senses TMA (TAAR5) in the physiologic regulation of circadian rhythms in mice.

Introduction

Modern investigation into human disease relies heavily on genetic and genomic approaches, and most assume that human disease is primarily driven by variation in the human genome. However, in the post-genomic era, we now understand that human genetic variation explains only a small proportion of risk for complex diseases such as obesity, diabetes, and cardiovascular disease (CVD). Instead, in many cases, environmental factors are the predominant drivers of cardiometabolic disease pathogenesis. Among all contributing environmental factors, it is clear that dietary patterns and diet-driven alterations in the gut microbiome can profoundly impact many common human diseases (Aron-Wisnewsky and Clément, 2016; Nobs et al., 2020; Arora and Bäckhed, 2016; Valles-Colomer et al., 2023). There are now many examples of diet–microbe–host interactions shaping human disease, but one of the more compelling is the reproducible link between elevated trimethylamine N-oxide (TMAO) levels and CVD risk (Wang et al., 2011; Tang et al., 2013; Koeth et al., 2013; Zhu et al., 2016; Skye et al., 2018; Bennett et al., 2013; Koeth et al., 2014; Leng et al., 2024). TMAO is generated by a metaorganismal (i.e. microbe and host) pathway where dietary substrates such as choline, l-carnitine, and γ-butyrobetaine are metabolized by gut microbial enzymes to generate the primary metabolite trimethylamine (TMA) (Wang et al., 2011; Tang et al., 2013; Koeth et al., 2013; Zhu et al., 2016; Skye et al., 2018; Bennett et al., 2013; Koeth et al., 2014; Leng et al., 2024). TMA is then further metabolized by the host enzyme flavin-containing monooxygenase 3 (FMO3) in the liver to produce TMAO (Bennett et al., 2013). Elevated TMAO levels are associated with many human diseases including diverse forms of CVD (Wang et al., 2011; Tang et al., 2013; Koeth et al., 2013; Zhu et al., 2016; Skye et al., 2018; Bennett et al., 2013; Koeth et al., 2014; Leng et al., 2024), obesity (Schugar et al., 2017; Dehghan et al., 2020), type 2 diabetes (Miao et al., 2015; Steinke et al., 2020), chronic kidney disease (CKD) (Tang et al., 2015; Zixin et al., 2022), neurodegenerative conditions including Parkinson’s and Alzheimer’s disease (Kumari et al., 2020; Vogt et al., 2018), and several cancers (Xu et al., 2015; Banerjee et al., 2024). Many of these disease associations have been validated in several large population meta-analyses (Li et al., 2022; Heianza et al., 2017; Schiattarella et al., 2017) and Mendelian randomization studies (Jia et al., 2019; Zhou et al., 2023). The emerging body of evidence supports the notion that elevated TMAO levels are causally related to cardiometabolic disease pathogenesis. In further support, therapies aimed at lowering circulating TMAO levels provide striking protection against cardiometabolic disease in animal models (Chen et al., 2019; Zhu et al., 2018; Wang et al., 2015; Roberts et al., 2018; Organ et al., 2020; Schugar et al., 2022; Helsley et al., 2022; Zhang et al., 2021; Gupta et al., 2020). Several independent studies have now shown that inhibition of either the host TMAO-producing enzyme FMO3 or the microbial TMA-producing enzyme CutC protects against diet-induced atherosclerosis (Miao et al., 2015; Wang et al., 2015), heart failure (Organ et al., 2020), thrombosis (Zhu et al., 2018; Roberts et al., 2018), obesity (Schugar et al., 2017; Schugar et al., 2022), liver disease (Helsley et al., 2022), insulin resistance (Schugar et al., 2017; Schugar et al., 2022), CKD (Zhang et al., 2021; Gupta et al., 2020), and abdominal aortic aneurysm (Benson et al., 2023).

Even though TMAO-lowering therapies are very effective in preclinical animal models, the underlying mechanism(s) linking the metaorganismal production of TMAO to cardiometabolic disease pathogenesis are still incompletely understood. Mechanistic understanding has been hampered by the fact that it is hard to disentangle the pleiotropic effects of dietary substrates (choline, carnitine, γ-butyrobetaine, trimethyllysine, etc.), bacterial production of TMA, and host-driven conversion of TMA to TMAO. At this point, the vast majority of studies have focused on the end product of this pathway, TMAO, but it is equally plausible that the primary metabolite, TMA, may play some role in microbe–host crosstalk related to human disease. There is some evidence that TMAO can promote inflammatory processes via activation of the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome, and nuclear factor κB (NFκB) (Seldin et al., 2016; Sun et al., 2016; Chen et al., 2017; Zhang et al., 2020). TMAO can also activate the endoplasmic reticulum (ER) stress kinase PERK (EIF2AK3) in hepatocytes to promote metabolic disturbance (Chen et al., 2019). In parallel, TMAO promotes stimulus-dependent calcium release in platelets to promote thrombosis (Zhu et al., 2016). Although the end product of the pathway TMAO clearly impacts cell signaling in the host to impact cardiometabolic disease, these TMAO-driven mechanisms do not fully explain how elevated TMAO levels contribute to so many diverse diseases in humans. In addition to TMAO-driven signaling mechanisms, we recently reported that major components of the TMAO pathway (choline, TMA, FMO3, and TAAR5) oscillate in a highly circadian fashion (Schugar et al., 2022). Furthermore, gut microbe-targeted drugs that selectively block TMA production alter host circadian rhythms in the gut microbiome and host phospholipid metabolism (Schugar et al., 2022). It is important to note that disruption of the circadian clock is a common hallmark of almost all diseases where TMAO levels are elevated (Sulli et al., 2018; Bolshette et al., 2023; Zheng et al., 2020; Bishehsari et al., 2020). To follow up on the potential links between the TMAO pathway and host circadian disruption, here we have used genetic knockout approaches at the level of host sensing of TMA (i.e. Taar5-/-), gut microbial TMA production (i.e. *cutC-*null microbial communities), and host TMA oxidation (i.e. Fmo3-/-). Results here further bolster the concept that TMA production and associated TAAR5 activation shapes host circadian rhythms.

Results

Mice lacking the TMA receptor TAAR5 have altered circadian rhythms in metabolic homeostasis and innate olfactory-related behaviors

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.

Host TAAR5 regulates the circadian rhythmicity of the gut microbiome

Bi-directional microbe–host communication is required for homeostatic control of chronobiology in the metaorganism (Mukherji et al., 2013; Asher and Sassone-Corsi, 2015; Thaiss et al., 2014; Liang et al., 2015; Choi et al., 2021). Our data strongly suggest that gut microbe-derived TMA can shape host circadian rhythms in metabolic homeostasis and behavior (Schugar et al., 2022; Massey et al., 2023), but we also wanted to test whether host TAAR5 may reciprocally regulate circadian rhythms of the gut microbiome (Thaiss et al., 2014; Liang et al., 2015). The rationale for studying alterations in the gut microbiome stems from the fact that several previous studies show that manipulating the TMAO pathway at different levels (diet, microbe, and host) strongly alters gut microbiota in unexpected ways (Koeth et al., 2013; Koeth et al., 2014; Roberts et al., 2018; Schugar et al., 2022). For example, provision of dietary substrates (i.e. choline or l-carnitine) or exogenous TMAO itself can reorganize gut microbial communities in mice (Koeth et al., 2013; Koeth et al., 2014). Also, small molecule enzyme inhibitors blocking the bacterial production of TMA profoundly alter the cecal microbiome (Roberts et al., 2018; Schugar et al., 2022), and some of the anti-obesity and circadian rhythm-altering effects can be transmitted by cecal microbial transplantation (Schugar et al., 2022). Therefore, we examined the circadian oscillation in the cecal microbiome in Taar5+/+ and Taar5-/- mice over a 24-hr period and found there were clear alterations that were time of day dependent (Figure 3, Figure 3—figure supplements 1 and 2; Supplementary file 1). At the phylum level, wild-type mice showed a modest decrease in the light cycle (i.e. ZT2–ZT10), and progressive loss of Firmicutes over the dark cycle (i.e. from ZT14–ZT22). In contrast, Taar5-/- mice showed a reciprocal increase during the light cycle and more modest dip during the light cycle in Firmicutes (Figure 3—figure supplement 1; Supplementary file 1). When we examined microbiome alterations at the genus level, there were much clearer alterations in specific bacteria (Figure 3; Figure 3—figure supplement 2; Supplementary file 1). In particular, the acrophase of several Lachnospiraceae, Odoribacter, and Dubosiella genera is altered in Taar5-/- mice when compared to wild-type controls (Figure 3; Figure 3—figure supplement 2; Supplementary file 1). Although there were many microbiome alterations, Taar5-/- mice showed delayed acrophase for Lachnospiraceae UCG-002, Lachnospiraceae NK4A136, Desulfovibrio, Bacteroides, Dubosiella, Colidextribacter, Alistipes, Turicibacter, Muribaculum, Helicobacter, and Parabacteroides, when compared to Taar5+/+ mice (Figure 3—figure supplement 2; Supplementary file 1). Other genera such as Lachnospiraceae UCG-006, Odoribacter, Butyricicoccus, Coriobacteriaceae UCG-002, Acetatifactor, and A2 showed advanced acrophase in Taar5-/- mice (Figure 3—figure supplement 2; Supplementary file 1). It is important to note that previous independent studies have also shown that either blocking bacterial TMA synthesis (Schugar et al., 2022) or FMO3-driven TMA oxidation (Zhu et al., 2018) can also strongly reorganize the gut microbiome in mice. Although more work is needed to fully understand the underlying mechanisms, it is clear that both gut microbe-driven TMA production and host sensing of TMA by TAAR5 (Figure 3; Figure 3—figure supplement 2; Supplementary file 1) can strongly impact circadian oscillations of the gut microbiome.

The trimethylamine receptor TAAR5 shapes circadian oscillations in the gut microbiome.

Male chow-fed wild-type (Taar5+/+) or mice lacking the TMA receptor (Taar5-/-) were necropsied at 4-hr intervals to collect cecum for microbiome composition analyses via sequencing the V4 region of the 16S rRNA (genus level changes are shown). (A) Canonical correspondence analysis (CCA) based beta diversity analyses show distinct microbiome compositions in Taar5+/+ and Taar5-/- mice. Statistical significance and beta dispersion were estimated using PERMANOVA. (B) The relative abundance of cecal microbiota in Taar5+/+ and Taar5-/- mice. Significantly altered cecal microbial genera in Taar5+/+ and Taar5-/- mice are shown at ZT2 (C), ZT6 (D), ZT10 (E), ZT14 (F), ZT18 (G), and ZT22 (H). ASVs that were significantly different in abundance (MetagenomeSeq with Benjamini–Hochberg false discovery rate (FDR) multiple test correction, adjusted p < 0.01). Data shown represent the means ± SD for n = 3–6 individual mice per group. Group differences were determined using ANOVA with Benjamini–Hochberg FDR multiple test correction, *adjusted p < 0.01.

Mice genetically lacking either gut microbial TMA production or host-driven TMA oxidation have altered circadian rhythms

To confirm and extend the idea that gut microbe-derived TMA can shape host circadian rhythms, we next performed experiments where we genetically deleted either gut microbial TMA synthesis or host-driven TMA oxidation. First, we used gnotobiotic mice engrafted with a defined microbial community with or without genetic deletion of the choline TMA lyase CutC (i.e. Clostridium sporogenes wild-type versus ΔcutC) to understand the ability of gut microbe-derived TMA to alter host circadian rhythms (Figure 4; Figure 4—figure supplement 1; Figure 4—figure supplement 2). Circadian oscillations in plasma TMA and TMAO that peak in the early dark cycle are only detectable in the group colonized with C. sporogenes (WT), but not ΔcutC C. sporogenes (Figure 4A). Other TMAO pathway-related metabolites, including choline, L-carnitine, γ-butyrobetaine, and betaine, also exhibited modest alterations in circadian rhythms (Figure 4A). Mice lacking choline TMA lyase activity showed modestly reduced mesor in plasma choline and advanced acrophase in both plasma betaine and γ-butyrobetaine (Figure 4A; Supplementary file 1). In a similar manner to Taar5-/- mice (Figure 1), mice colonized with ΔcutC C. sporogenes show alterations in the expression of some but not all circadian genes in the olfactory bulb (Figure 4B; Supplementary file 1). Mice colonized with ΔcutC C. sporogenes had significantly advanced acrophase for Bmal1, yet delayed acrophase for Clock, in the olfactory bulb when compared to mice colonized with the control community (Figure 4B; Supplementary file 1). However, the oscillatory pattern of other circadian genes was not significantly altered. When we examined circulating hormones and cytokines, there were clear alterations in the circadian oscillation in mice genetically lacking choline TMA lyase activity (Figure 4C; Supplementary file 1). Mice colonized with the ΔcutC C. sporogenes community exhibited delayed acrophase for plasma insulin and interleukins 2 (IL-2) and 33 (IL-33) when compared to mice harboring the wild-type community that could produce TMA (Figure 4C; Supplementary file 1). Mice lacking choline TMA lyase activity also had an advanced acrophase for GLP-1, leptin, and IL-1β (Figure 2C; Supplementary file 1). It is interesting to note that we examined the cecal abundance of the five bacterial strains in our defined community. There were clear alterations that were time of day dependent (Figure 4—figure supplement 1). Four of the bacterial strains (C. sporogenes, B. theta, B. caccae, and B. ovatus) showed clear circadian oscillations when a zero amplitude test was performed in mice colonized with wild-type community. However, the community lacking cutC lost all significant circadian oscillation of these same four bacterial strains (Figure 4—figure supplement 1; Supplementary file 1), showing that bacterial production of TMA can broadly impact both microbe–microbe and microbe–host interactions in circadian rhythms.

Transplanting a defined synthetic microbial community with or without genetically deleted trimethylamine production capacity (ΔcutC) alters host circadian rhythms.

Germ-free C57Bl/6 mice (recipients) were gavaged with the core community (B. caccae, B. ovatus, B. thetaiotaomicron, C. aerofaciens, and E. rectale) with TMA producing wild-type (WT) C. sporogenes (produces TMAO) or C. sporogenes ΔcutC. Gnotobiotic mice were then necropsied at 4-hr intervals to collect tissues including plasma (A, C) and olfactory bulb (B). (A) Plasma levels of TMAO pathway metabolites (choline, L-carnitine, betaine, γ-butyrobetaine, trimethylamine (TMA), and trimethylamine N-oxide (TMAO)) were quantified by liquid chromatography–tandem mass spectrometry (LC–MS/MS). (B) PCR was performed on olfactory bulb to examine key circadian clock regulators. (C) Plasma levels of metabolic hormones (insulin, GLP-1, and leptin) and select cytokines including interleukins (IL-1β, IL-2, and IL-33) were measured as described in the Methods section. Data shown represent the means ± SEM for n = 5–6 individual mice per group. Differences between WT-cutC and ΔcutC groups were determined using cosinor analyses, and p-values are provided where there were statistically significant differences between groups for circadian statistics. The complete cosinor statistical analysis for circadian data can be found in Supplementary file 1. Significant differences between WT-cutC and ΔcutC groups were also analyzed by Student’s t-tests within each ZT time point (*p < 0.05 and **p < 0.01).

To follow up on our previous findings that FMO3 suppresses both beige and brown fat-induced cold-induced thermogenesis (Schugar et al., 2017), we also wanted to examine circadian patterns in subscapular BAT of ΔcutC C. sporogenes-colonized mice (Figure 4—figure supplement 2; Supplementary file 1). Although there were no statistically significant alterations in circadian gene expression in the BAT isolated from ΔcutC C. sporogenes-colonized mice, the acrophase of phosphatidylethanolamine methyltransferase (Pemt) was delayed in mice colonized with the ΔcutC community compared to mice colonized with the control wild-type community (Figure 4—figure supplement 2A; Supplementary file 1). We next wanted to more comprehensively quantify a variety of other well-known metabolites that are known to originate from bacterial sources (Figure 4—figure supplement 2B). The rationale for performing microbe-focused metabolomics is that we have found if we alter one gut microbe-derived metabolite in other gnotobiotic mouse studies, there can be unexpected alterations in other distinct classes of microbe-derived metabolites (Wang et al., 2023). This is likely due to the fact that complex microbe–microbe and microbe–host interactions work together to define systemic levels of circulating metabolites, influencing both the production and turnover of distinct and unrelated metabolites. Although the targeted deletion of cutC in C. sporogenes prevents the production of TMA from choline, there were alterations of several other gut microbe-derived metabolites in mice colonized with the ΔcutC C. sporogenes community (Figure 4—figure supplement 2). Bacterially derived aromatic amino acid metabolites such as hippuric acid, indole-3-propionic acid, and indole acetic acid were significantly reduced in mice colonized with the ΔcutC community (Figure 4—figure supplement 2). Also, the bacterially derived phenylalanine metabolites phenylacetic acid and phenylacetylglycine gained rhythmicity with marked increases in their peak amplitude in mice colonized with the ΔcutC community when compared to control mice (Figure 4—figure supplement 2).

Finally, we wanted to examine whether host co-metabolism of TMA can also alter circadian rhythms (Figure 4—figure supplement 2). Although gut microbes are the sole source of TMA, circulating levels are also shaped by abundant conversion of TMA to TMAO by the host liver enzyme FMO3 (Schugar et al., 2017; Zhu et al., 2018). To test whether the hepatic conversion of TMA to TMAO by FMO3 may alter circadian rhythms, we necropsied female Fmo3+/+ and Fmo3-/- mice at ZT2 (early light cycle) or ZT14 (early dark cycle). Fmo3-/- mice have reduced levels of TMAO at both ZT2 and ZT14, as well as reduced expression of key circadian genes Bmal1 and Per1 at ZT2 (Figure 4—figure supplement 2). Taken together, our data suggest that bacterial production, FMO3-driven metabolism of TMA, as well as sensing of TMA by the host receptor TAAR5, converge to shape circadian rhythms in gene expression, metabolic hormones, gut microbiome composition, and innate behaviors.

Discussion

The metaorganismal TMAO pathway is strongly associated with many human diseases (Wang et al., 2011; Tang et al., 2013; Koeth et al., 2013; Zhu et al., 2016; Skye et al., 2018; Bennett et al., 2013; Koeth et al., 2014; Leng et al., 2024; Schugar et al., 2017; Dehghan et al., 2020; Miao et al., 2015; Steinke et al., 2020; Tang et al., 2015; Zixin et al., 2022; Kumari et al., 2020; Vogt et al., 2018; Xu et al., 2015; Banerjee et al., 2024; Li et al., 2022; Heianza et al., 2017; Schiattarella et al., 2017; Jia et al., 2019; Zhou et al., 2023; Chen et al., 2019; Zhu et al., 2018), which has prompted the rapid development of drugs intended to lower circulating levels of TMAO (Wang et al., 2015; Roberts et al., 2018; Organ et al., 2020; Schugar et al., 2022; Helsley et al., 2022; Zhang et al., 2021; Gupta et al., 2020; Benson et al., 2023). As the rapid drug discovery advances toward human studies, it will be extremely important to understand the diverse mechanisms by which the primary metabolite TMA and/or the secondary metabolite TMAO promotes disease pathogenesis in the human metaorganism. There is compelling evidence that the end product of the pathway TMAO can promote inflammation, ER stress, and platelet activation via activation of NFκB, the NLRP3 inflammasome, PERK, and stimulus-dependent calcium release, respectively (Chen et al., 2019; Seldin et al., 2016; Sun et al., 2016; Chen et al., 2017; Zhang et al., 2020). However, these TMAO-driven mechanisms only partially explain the links to so many diverse human diseases. Here, we provide new evidence that in addition to these TMAO-driven mechanisms, the primary gut microbe TMA can in parallel shape circadian rhythms through the host GPCR TAAR5. The major findings of the current studies are: (1) Mice lacking the TMA receptor TAAR5 have abnormal oscillations in core circadian genes, particularly in the olfactory bulb, (2) Compared to wild-type controls, Taar5-/- mice have altered circulating levels of cytokines, metabolic hormones, and metabolites at certain times of the day, (3) Taar5-/- mice have altered innate and repetitive behaviors that emerge only in a time of day-dependent manner, (4) The normal oscillatory behavior of the cecal microbiome is dysregulated in Taar5-/- mice, (5) Genetic deletion of gut microbial choline TMA lyase activity, using defined cutC-null microbial communities in vivo, results in rewiring of circadian rhythms in gene expression, metabolic hormones, cytokines, and metabolites, (6) The bacterial strains in the defined microbial used in gnotobiotic mouse study oscillate differently depending on the presence of cutC, and (7) Mice lacking the host liver TMA-to-TMAO converting enzyme FMO3 likewise have altered circadian gene expression in the olfactory bulb (Figure 5). Collectively, our findings suggest that therapeutic strategies designed to limit gut microbial TMA production (i.e. TMA lyase inhibitors), host liver TMA oxidation (i.e. FMO3 inhibitors), or sensing of TMA by the host GPCR TAAR5 (i.e. TAAR5 inhibitors) will need to be carefully evaluated for pleiotropic effects on circadian rhythms. This body of work also demonstrates that diet–microbe–host interactions can powerfully shape chronobiology and provides one of the first examples of a microbial metabolite being sensed by a host GPCR to rewire circadian rhythms.

Summary of findings.

Dietary choline is converted by gut microbial CutC/D into TMA, which signals through the host receptor TAAR5 or is converted to TMAO by hepatic FMO3. Loss of TAAR5 disrupts core circadian gene oscillations (particularly in the olfactory bulb), alters time-of-day regulation of cytokines, hormones, metabolites, and reveals time-dependent changes in innate and repetitive behaviors, alongside dysregulated oscillatory microbiome dynamics. Eliminating microbial cutC similarly rewires circadian oscillations in host immune and metabolic pathways, and microbial strains themselves exhibit altered rhythmicity depending on cutC status. Likewise, Fmo3⁻/⁻ mice display disturbed circadian gene rhythms, together defining a microbial TMA–TAAR5–FMO3 axis as a key regulator of circadian control, inflammation, and metabolic disease-relevant physiology.

Currently, the only known host receptor that senses gut microbe-produced TMA is the volatile amine receptor TAAR5, which allows for species-specific recognition of the ‘fishy’ odor intrinsic to TMA but not TMAO (Wallrabenstein et al., 2013; Li et al., 2013; Liberles, 2015). In general, the trace amine-associated receptor (TAAR) subfamily of GPCRs primarily function as olfactory receptors in vertebrates (Liberles, 2015; Freyberg and Saavedra, 2020). Ligand-dependent activation of each individual TAAR receptor occurs in a unique sensory neuron population primarily localized to olfactory cilia, and when activated typically elicits TAAR-specific olfactory-related behavior responses (Liberles, 2015; Freyberg and Saavedra, 2020). TMA-dependent activation of TAAR5 was first shown to promote attraction and social interaction between females and male mice in a very species and strain-specific manner (Li et al., 2013). This seminal study by Liberles and colleagues showed that the TMA–TAAR5 olfactory circuit can powerfully shape olfactory-driven social interaction, and FMO3-driven conversion of TMA to TMAO suppresses TAAR5 activation (Li et al., 2013). In parallel, we recently performed a comprehensive study to assess cognitive, motor, anxiolytic, social, olfactory, and innate behaviors in mice treated with small molecule choline TMA lyase inhibitors to block bacterial TMA production (Massey et al., 2023). Much like the olfactory and social phenotypes seen in Taar5-/- mice (Li et al., 2013), we found that pharmacologic blockade of bacterial TMA production significantly altered olfaction and olfactory-related social behaviors, but did not significantly alter cognitive, motor, and anxiolytic behaviors (Massey et al., 2023). It is interesting to note that choline TMA lyase inhibitors altered olfactory perception of several odorant stimuli beyond TMA itself including a cookie, almond, vanilla, corn oil, and coyote urine (Massey et al., 2023). This indicates that drugs blocking TMA–TAAR5 signaling could impact olfactory neurogenesis to impact sensing of diverse odorant cues that are well beyond the rotten fish smell intrinsic to TMA. Given that anosmia is a common occurrence in several TMAO-related diseases including