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 and metabolism related genes was quantified by qPCR. 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 S1. * = Significant differences between Taar5+/+ andTaar5-/- mice by student’s t-tests within each ZT time point (p<0.05).

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 hormones and cytokine levels were quantified as described in the material 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 S1. * = Significant differences between Taar5+/+ andTaar5-/- mice by student’s t-tests within each ZT time point (p<0.05).

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., and statistically significant difference between Taar5+/+ and Taar5-/- mice are denoted by * = p<0.05 and ** = p<0.01.

Mice Lacking the Host TMA Receptor TAAR5 Have Altered Behaviors.

Male or female wild type mice (Taar5+/+) or mice lacking the TMA receptor (Taar5-/-) were subjected to a battery of behavioral test including: (A) startle test, (B) forepaw grip strength test, (C) hotplate sensitivity test, (D) rotorod test, (E) social interaction with a juvenile test, with initial testing and then again three days later. (F) fear conditioning cue test, (G) elevated plus maze, (H) prepulse inhibition, (I) Y-maze, and (J) indirect calorimetry using the Oxymax CLAM metabolic cage system. 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., 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).

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. The complete cosinor statistical analysis for circadian data can be found in Table S1. * = Significant differences between Taar5+/+ andTaar5-/- mice by student’s t-tests within each ZT time point (p<0.05).

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 choline, L-carnitine, betaine, ψ-butyrobetaine, trimethylamine (TMA), and trimethylamine N-oxide (TMAO) were quantified by liquid chromatography with online tandem mass spectrometry (LC-MS/MS). (B) PCR was performed on olfactory bulb to examine key circadian clock regulators. (C) Plasma levels of select cytokines including interleukins (IL-1b, IL-2, and IL-33) and other plasma metabolites and hormones 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 S1. 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 cytokines and metabolite levels were quantified as described in the method section. Data for panels B and C were analyzed by cosinor analyses and representative p-values are shown; The complete statistical analysis for circadian metrics can be found in Table S1. (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. Group differences 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 S1. Significant differences between by student’s t-tests within each ZT time point (* = p<0.05 and ** = p<0.01).

Cosinor analyses for all circadian data include in this manuscript.