The Mouse Microbiome Is Required for Sex-Specific Diurnal Rhythms of Gene Expression and Metabolism

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https://www.cell.com/cell-metabolism/fulltext/S1550-4131(18)30631-4?dgcid=raven_jbs_etoc_email

The Mouse Microbiome Is Required for Sex-Specific Diurnal Rhythms of Gene Expression and Metabolism
Summary
The circadian clock and associated feeding rhythms have a profound impact on metabolism and the gut microbiome. To what extent microbiota reciprocally affect daily rhythms of physiology in the host remains elusive. Here, we analyzed transcriptome and metabolome profiles of male and female germ-free mice. While mRNA expression of circadian clock genes revealed subtle changes in liver, intestine, and white adipose tissue, germ-free mice showed considerably altered expression of genes associated with rhythmic physiology. Strikingly, the absence of the microbiome attenuated liver sexual dimorphism and sex-specific rhythmicity. The resulting feminization of male and masculinization of female germ-free animals is likely caused by altered sexual development and growth hormone secretion, associated with differential activation of xenobiotic receptors. This defines a novel mechanism by which the microbiome regulates host metabolism.

Introduction
Obesity-related metabolic diseases cause major public health problems. Dysregulation of the microbiome (
Winer et al., 2016
) and the circadian clock have been implicated in the progression of these diseases (
Laermans and Depoortere, 2016
). Many physiological processes, including fatty acid (FA) and bile acid (BA) metabolism, as well as cytokine and corticosteroid secretion, are regulated by both circadian rhythms and microbiota. Remarkably, the composition and metabolic activity of commensal bacteria show diurnal variations that depend on the circadian clock (
Liang et al., 2015
,
Thaiss et al., 2014
,
Voigt et al., 2016
), feeding rhythms (
Thaiss et al., 2014
,
Thaiss et al., 2016
,
Voigt et al., 2014
,
Zarrinpar et al., 2014
), and the nutritional composition of the diet (
Leone et al., 2015
,
Voigt et al., 2014
,
Zarrinpar et al., 2014
). Vice versa, microbiota can feed back on clock gene expression in the host. Indeed, germ-free (GF) or antibiotic-treated mice show disturbed circadian clock gene expression in liver and intestine (
Björkholm et al., 2009
,
Joyce et al., 2014
,
Leone et al., 2015
,
Montagner et al., 2016
,
Mukherji et al., 2013
). Moreover, conditions altering the composition of the microbiome, such as high-fat diet (HFD) (
Hildebrandt et al., 2009
), obesity (
Ley et al., 2005
), and Roux-en-Y gastric bypass surgery (
Tremaroli et al., 2015
) also affect host circadian clock gene expression (
Ando et al., 2011
,
Kim et al., 2015
,
Kohsaka et al., 2007
). However, effects of microbiome on the host circadian clock in peripheral tissues show striking inconsistencies.
To further study the relationship between the host-microbiota relationship and the circadian clock, we used RNA sequencing (RNA-seq) of conventionally raised (ConvR) and GF mice to compare temporal gene expression in liver, duodenum, ileum, and perigonadal white adipose tissue (WAT). We found that the molecular clock was globally unaffected, but genes involved in key metabolic processes exhibited an altered rhythmic expression in GF mice. Strikingly, we observed a feminization of gene expression (i.e., downregulation of male-biased and upregulation of female-biased genes) in the liver and WAT of GF male mice. In females, our analysis confirmed an attenuation of sexually dimorphic rhythmic gene expression and metabolic activities in GF mice. This was a consequence of altered sex-hormone and growth hormone (GH) signaling, likely due to the defective sexual maturation of GF mice. These results highlight the key role of the microbiota on the establishment of sexually dimorphic liver metabolism and tentatively elucidate several unexplained phenotypes of GF mice that are also known to be sexually dimorphic, such as resistance to liver cancer

Results
Global Alteration of Gene Expression in the Digestive Tract of GF Mice
To study the impact of microbiota on host gene expression, we compared temporal gene expression in the liver, duodenum, ileum, and WAT of GF and ConvR male mice (Figure S1A). The transcriptomes of GF and ConvR could be distinguished in the liver, duodenum, and ileum but less so in WAT (Figure S1B). We first analyzed constitutive changes between GF and ConvR mice in the different tissues. In the duodenum and ileum of GF mice, most affected genes displayed a significant decrease in expression (Figure S1C and Table S1). Specifically, genes associated with immune response and immune cell mobility were downregulated in the intestine of GF mice (Figure S1D). In the liver, we detected a significant alteration for genes involved in lipid and cholesterol metabolism, confirming the role of microbiota in these pathways (Joyce et al., 2014). In WAT, genes related to immune response were downregulated, consistent with previous reports (Caesar et al., 2015).

Microbiota Depletion Alters Rhythmic Gene Expression
Next, we quantified rhythmic gene expression in ConvR and GF mice (see STAR Methods and (Atger et al., 2015)) (Figure 1A and Table S1). The analysis revealed that most rhythmic transcripts are unaffected (model 4). Interestingly, a higher proportion of genes gained rather than lost rhythmicity in the duodenum and ileum of GF mice (model 2). In the liver and WAT, this ratio was reversed (Figures 1B and 1C). Circadian clock and clock output genes showed only subtle changes in phase and amplitude of mRNA abundance across tissues (Figures 1D and S1E–S1H). Most of the genes that lost rhythmicity in the duodenum of GF mice were associated with ribosome biogenesis, a rhythmically orchestrated process in the liver (Sinturel et al., 2017,Wang et al., 2017), and glutamine metabolism, reported to correlate with colonization of the intestine (
El Aidy et al., 2013) (Table S2). In the ileum, the genes that lost rhythmicity were involved in the organization of the extracellular matrix, potentially reflecting its rhythmic regeneration after its degradation by contacts with bacteria (
Thaiss et al., 2016
). Moreover, genes involved in carbohydrate metabolism gained rhythmicity in the ileum of GF mice, suggesting that microbiota, known to regulate glucose transport and metabolism (
Donohoe et al., 2012
), dampen rhythmic glucose metabolism in ConvR mice. In WAT of GF mice, the loss of rhythmicity in genes involved in cytokine signaling might reflect microbiota-dependent rhythmic inflammatory processes (
Caesar et al., 2015
). Meanwhile, genes associated with deoxyribonucleotide metabolism, a process showing increased activity during gut colonization (
El Aidy et al., 2013
), gained rhythmicity in GF mice.