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-hour 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 (Arntl, 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 -/+ S.D. for n= 3-6 individual mice per group. Group differences were determined using cosinor analyses p-values are provided where there were statistically significant differences between Taar5+/+ andTaar5-/- mice. The complete cosinor statistical analysis for circadian data can be found in Table 1. * = Significant differences between Taar5+/+ andTaar5-/- mice by student’s t-tests within each ZT time point (p<0.05).

Cosinor statistics for all circadian data include in this manuscript.

Mice Lacking the Host TMA Receptor TAAR5 Have Altered Olfactory and Repetitive Behaviors Only at Specific Circadian Timepoints.

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 -/+ S.E.M. 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.

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-hour 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+/+ andTaar5-/- 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 FDR multiple test correction, adjusted p<0.01). Data shown represent the means -/+ S.D. for n= 3-6 individual mice per group. Group differences were determined using ANOVA with Benjamini-Hochberg false discovery rate (FDR) multiple test correction, * = adjusted p<0.01.

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, E. rectale) with TMA producing wild-type (WT) C. sporogenes (produces TMAO) or C. sporogenes ΔcutC. Gnotobiotic mice were then necropsied at 4-hour intervals to collect tissues including plasma (A and 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 -/+ S.E.M. for n= 5-6 individual mice per group. Differences between WT-cutC and ΔcutC groups were determined using cosinor analyses p-values are provided where there were statistically significant differences between group for circadian statistics. The complete cosinor statistical analysis for circadian data can be found in Table 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).

Body Weights Across a 24-Hour Period in Chow-Fed Male Taar5+/+ and Taar5-/- Mice.

Male chow-fed wild-type (Taar5+/+) or mice lacking the TMA receptor (Taar5-/-) were necropsied at 4-hour intervals and body weight at necropsy was quantified. Data shown represent the means -/+ S.D. for n= 3-6 individual mice per group. Group differences were tested using cosinor analyses, but no statistically-signficant differences were detected.

The Host Trimethylamine Receptor TAAR5 Shapes Circadian Oscillations in Circulating Hormones and Cytokines.

Male chow-fed wild-type mice (Taar5+/+) or mice lacking the TMA receptor (Taar5-/-) were necropsied at 4-hour intervals to collect plasma. (A) Plasma levels of metabolites in the metaorganismal trimethylamine N-oxide (TMAO) pathway were quantified using liquid chromatography tandem mass spectrometry (LC-MS/MS). (B) Plasma metabolic hormones (insulin, C-peptide, glucagon, GLP-1, leptin, ghrelin, and peptide YY) and cytokine/chemokine (MCP-1, IL-6, and TNFα) levels were quantified using MesoScale Discovery multi-plex immunoassays as described in the materials and methods. Data shown represent the means -/+ S.D. for n= 3-6 individual mice per group. Group differences were determined using cosinor analyses p-values are provided where there were statistically significant differences between Taar5+/+ andTaar5-/- mice. The complete cosinor statistical analysis for circadian data can be found in Table 1. Significant differences between Taar5+/+ andTaar5-/- mice by student’s t-tests within each ZT time point (*p< 0.05: **p< 0.01; ***p <0.001, and ****p<0.0005)

Olfactory Discrimination of Several Odor Stimuli is Unaltered in Taar5-Deficient Mice.

Male or female wild-type mice (Taar5+/+) or mice lacking the TMA receptor (Taar5-/-) were subjected to a battery of single stimulus olfactory discrimination tests to determine time to and time at each stimuli including: (A) banana, (B) corn oil, (C) almond, (D) water, (E) or social odors as described in the methods section. Data are shown for the entire cohort combining both sexes or divided into either male or female cohorts to examine sexual dimorphism in phenotype. Data represent the mean -/+ S.E.M. from n=10-15 per group when male and female are separated (n=25-27 when both sexes are combined). No statistically significant alterations were found during this battery of tests.

Taar5-Deficient Mice Exhibit Specific Alterations in Social Behaviors.

Male or female wild-type mice (Taar5+/+) or mice lacking the TMA receptor (Taar5-/) were subjected to a battery of social behavioral tests including: (A) three-chamber preference test, (B) three-chamber social preference test, (C) three-chamber social novelty test, or (D) social interaction with a juvenile as described in the methods section. Data are shown for the entire cohort combining both sexes or divided into either male or female cohorts to examine sexual dimorphism in phenotype. Data represent the mean -/+ S.E.M. from n=10-15 per group when male and female are separated (n=25-27 when both sexes are combined), Significant differences between Taar5+/+ and Taar5-/- mice were determined by student’s t-tests (*p< 0.05: **p< 0.01; ***p <0.001, and ****p<0.0005)

Taar5-Deficient Mice Exhibit Specific Alterations in Innate Behavioral Responses.

Male or female wild-type mice (Taar5+/+) or mice lacking the TMA receptor (Taar5-/) were subjected to a battery of innate behavioral tests including: (A) startle test, (B) forepaw grip strength, (C) hotplate sensitivity, (D) rotorod, and (E) nesting as described in the methods section. Data are shown for the entire cohort combining both sexes or divided into either male or female cohorts to examine sexual dimorphism in phenotype. Data represent the mean -/+ S.E.M. from n=10-15 per group when male and female are separated (n=25-27 when both sexes are combined). Significant differences between Taar5+/+ and Taar5-/- mice were determined by student’s t-tests (*p< 0.05: **p< 0.01; and ***p <0.001).

Impact of Taar5-Deficiency on Cognitive, Depression, and Anxiety-Like Behaviors.

Male or female wild-type mice (Taar5+/+) or mice lacking the TMA receptor (Taar5-/) were subjected to a battery of behavioral tests related to cognition, depression, and anxiety including: (A) cued fear conditioning, (B) elevated plus maze, (C) Y-maze, and (D) open field tes as described in the methods section. Data are shown for the entire cohort combining both sexes or divided into either male or female cohorts to examine sexual dimorphism in phenotype. Data represent the mean -/+ S.E.M. from n=9-10 per group when male and female are separated (n=19-20 when both sexes are combined). Significant differences between Taar5+/+ and Taar5-/- mice were determined by student’s t-tests (*p< 0.05)

Impact of Taar5-Deficiency on Morris Water Maze Performance.

Male or female wild-type mice (Taar5+/+) or mice lacking the TMA receptor (Taar5-/) were subjected to the Morris water maze as described in the methods section. Data are shown for the entire cohort combining both sexes or divided into either male or female cohorts to examine sexual dimorphism in phenotype. Data represent the mean -/+ S.E.M. from n=10-15 per group when male and female are separated (n=25-27 when both sexes are combined), Significant differences between Taar5+/+ and Taar5-/- mice were determined by student’s t-tests (*p< 0.05: **p< 0.01; ***p <0.001, and ****p<0.0005)

Impact of Taar5-Deficiency on Systemic Energy Metabolism and Gene Expression in Brown Adipose Tissue (BAT).

(A) Male or female wild-type mice (Taar5+/+) or mice lacking the TMA receptor (Taar5-/) were place in individual cages for indirect calorimetry measured using the Oxymax CLAMS home cage system. After two days of equillbration, mice were maintained at thermoneutrality (30°C), room temperature (22°C), or cold stressed (4°C) over a 24-hour period at each temperature point. Oxygen consumption was quantified throughout these temperature transitions as described in the methods section. (B) Male or female wild-type mice (Taar5+/+) or mice lacking the TMA receptor (Taar5-/) were necropsied at the beginning of the light cycle (ZT2) or the beginning of the dark cycle (ZT14) and subscapular brown adipose tissue (BAT) was harvested to examine gene. The relative gene expression for circadian genes (Arntl, Nr1d1, Cry1, and Per1) was quantified by qPCR using the ΔΔ-CT method. Data represent the mean -/+ S.E.M. from n=5-9 per group. Significant differences between Taar5+/+ and Taar5-/- mice were determined by student’s t-tests within each individual time point (*p< 0.05)

TAAR5-Deficient Mice Have Altered Circadian Oscillations in the Gut Microbiome at the Phylum Level.

Male chow-fed wild-type (Taar5+/+) or mice lacking the TMA receptor (Taar5-/-) were necropsied at 4-hour intervals to collect cecum for microbiome composition analyses via sequencing the V4 region of the 16S rRNA (phylum level changes are shown). Data shown represent the means -/+ S.D. for n= 3-6 individual mice per group. The relative abundance of cecal microbiota at the phylum level are shown, and group differences were determined using cosinor analyses p-values are provided where there were statistically significant differences between Taar5+/+ andTaar5-/- mice.

TAAR5-Deficient Mice Have Altered Circadian Oscillations in the Gut Microbiome.

Male chow-fed wild-type (Taar5+/+) or mice lacking the TMA receptor (Taar5-/-) were necropsied at 4-hour intervals to collect cecum for microbiome composition analyses via sequencing the V4 region of the 16S rRNA (genus level changes are shown). The relative abundance of cecal microbiota at the genus level are shown, and group differences were determined using cosinor analyses p-values are provided where there were statistically significant differences betweenTaar5+/+ andTaar5-/- mice. Data shown represent the means -/+ S.D. for n= 3-6 individual mice per group. Group differences were tested using cosinor analyses (key statistics represented here), and the complete cosinor statistical analysis for circadian data can be found in Table 1. * = Significant differences are also shown between Taar5+/+ andTaar5-/- mice by student’s t-tests within each ZT time point (p<0.05).

The Oscillatory Patterns of the TMAO-Defined Community is Altered When cutC is Genetically Deleted.

Germ-free C57Bl/6 mice (recipients) were gavaged with the core community (B. caccae, B. ovatus, B. thetaiotaomicron, C. aerofaciens, E. rectale) with TMA producing wild-type (WT) C. sporogenes (produces TMAO) or C. sporogenes ΔcutC. Gnotobiotic mice were then necropsied at 4-hour intervals to collect cecum for shotgun metagenomic sequencing. The total abundance is shown for each of the five bacteria represented in the defined community over the 24-hour circadian period. Differences between WT-cutC and ΔcutC groups were determined using cosinor analyses p-values are provided where there were statistically significant differences between group for circadian statistics. The complete cosinor statistical analysis for circadian data can be found in Table 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).

Mice Lacking Either Gut Microbial TMA Production or Host-Driven TMA Oxidation Have Altered Circadian Rhythms.

(A,B) Female germ-free C57Bl/6 mice (recipients) were gavaged with the core community (B. caccae, B. ovatus, B. thetaiotaomicron, C. aerofaciens, E. rectale) with TMA producing wild-type (WT) C. sporogenes (produces TMAO) or C. sporogenes ΔcutC. Gnotobiotic mice were then necropsied at 4-hour intervals to collect tissues including subscapular brown adipose tissue (A) and plasma (B). Gene expression was quantified by qPCR and metabolite levels were quantified by LC-MS/MS as described in the method section. Data for panels A and B were analyzed by cosinor analyses and representative p-values are shown; The complete statistical analysis for cosinor circadian metrics can be found in Table 1. (C) Female wild-type (Fmo3+/+) or flavin-containing monooxygenase 3 knockout (Fmo3-/-) mice were necropsied at ZT2 or ZT14 and TMAO levels were measured by LC-MS/MS and olfactory bulb circadian gene expression was quantified by qPCR. Data shown represent the means -/+ S.E.M. for n= 4-6 individual mice per group. Significant differences between by student’s t-tests within each ZT time point (* = p<0.05 and ** = p<0.01).