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The intestinal microbiota regulates body composition through NFIL3 and the circadian clock

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Science  01 Sep 2017:
Vol. 357, Issue 6354, pp. 912-916
DOI: 10.1126/science.aan0677
  • Fig. 1 Nfil3ΔIEC mice are resistant to high-fat diet–induced obesity.

    (A) qRT-PCR analysis of Nfil3 transcript abundance in small intestinal epithelial cells recovered by laser capture microdissection from conventional (conv) and germ-free mice. (B) Age-matched Nfil3fl/fl and Nfil3ΔIEC mice were cohoused and placed on a high-fat diet (HFD) for 10 weeks. Body weight was measured before and after diet switching. (C) Body-fat percentages of mice in (B). (D) Epididymal fat-pad weight. (E) Serum triglyceride concentration. (F) Hematoxylin and eosin (H&E) staining of liver (scale bars, 100 μm). (G) Glucose-tolerance and insulin-tolerance tests. (H) Body-fat percentage of mice treated with or without antibiotics after switching to a HFD. All data represent two independent experiments with four to eight mice per group. Male mice were used in all experiments. Means ± SEM (error bars) are plotted; statistics were performed with Student’s t test or one-way analysis of variance (ANOVA). *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

  • Fig. 2 The microbiota induces epithelial NFIL3 expression through the circadian clock factor REV-ERBα and a DC-ILC3 signaling relay.

    (A to D) qRT-PCR analysis of Nfil3 (A) and Rev-erbα (C) transcript abundance in small intestinal epithelial cells from germ-free (dotted line) and conventional (solid line) mice across a 24-hour day-night light cycle. Western blot analysis of NFIL3 (B) and REV-ERBα (D) was performed on small intestinal epithelial cells isolated from conventional or germ-free mice. Lamin B was used as the loading control. ZT, Zeitgeber time. (E) Chromatin immunoprecipitation assay on intestinal epithelial cells using immunoglobulin G (IgG) or an antibody to REV-ERBα (anti-REV-ERBα). Precipitated fragments of the Nfil3 promoter were detected by qRT-PCR. (F) qRT-PCR analysis of epithelial Nfil3 expression in conventional (conv) wild-type (wt), antibiotic (Abx)–treated wild-type, or Abx-treated Rev-erbα−/− mice. (G) qRT-PCR analysis of epithelial Rev-erbα and Nfil3 expression in germ-free and conventional wild-type mice and conventional Myd88fl/fl, Myd88−/−, Myd88ΔIEC (epithelial cell–specific knockout), and Myd88ΔDC (DC-specific knockout) mice. (H) qRT-PCR analysis of epithelial Rev-erbα and Nfil3 expression in germ-free and conventional wild-type mice, conventional Cd11c-DTR mice that were untreated or treated with Diphtheria toxin (DT), Id2gfp/gfp mice, and Rag1−/− mice. (I and J) qRT-PCR analysis of epithelial Nfil3 (I) and Rev-erbα (J) expression in Rorc+/+ (solid line) and Rorcgfp/gfp (dotted line) mice. (K) qRT-PCR analysis of epithelial Rev-erbα and Nfil3 expression in Myd88−/− mice treated with recombinant IL-23, IL-22, or vehicle. Data in (E), (F), (G), (H), and (K) were collected at ZT4. N = 3 to 8 mice per group. Means ± SEM (error bars) are plotted; statistics were performed with Student’s t test or one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

  • Fig. 3 STAT3 represses Rev-erbα transcription by binding directly to its promoter.

    (A) Schematic of the Rev-erbα gene promoter. (B) ChIP analysis of intestinal epithelial cells from conventional or germ-free mice using IgG or anti-STAT3 antibody. Precipitated fragments of the Rev-erbα promoter or control exon were detected by qRT-PCR. (C) Luciferase reporter assay. A 504-bp fragment of the Rev-erbα promoter was fused to a firefly luciferase reporter. HEK-293T cells were transfected with reporters and empty vector, a wild-type STAT3-encoding vector (Stat3-wt), or a dominant active STAT3-encoding vector (Stat3-c). RLU, relative light units. (D) Western-blot of total STAT3 and phosphorylated STAT3 (p-STAT3) in small intestinal organoids treated with IL-22 and/or the STAT3 inhibitor Stattic. Mito 70 was used as the loading control. (E and F) qRT-PCR analysis of Rev-erbα (E) and Nfil3 (F) expression in small intestinal organoids treated with IL-22 and/or Stattic. (G and H) qRT-PCR analysis of epithelial Rev-erbα (G) and Nfil3 (H) expression in Stat3fl/fl and Stat3ΔIEC mice at ZT4. N = 3 to 8 samples per group. Means ± SEM (error bars) are plotted; statistics were performed with Student’s t test or one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.

  • Fig. 4 Epithelial NFIL3 controls expression of a circadian lipid metabolic program and regulates lipid absorption in intestinal epithelial cells.

    (A) RNA sequencing analysis of epithelial cell transcripts in Nfil3fl/fl and Nfil3ΔIEC mice across a circadian cycle. The heat map displays expression levels of the 33 genes that have altered expression in Nfil3ΔIEC mice as compared with Nfil3fl/fl mice. Genes encoding proteins that function in lipid metabolism are highlighted in blue. Top panels show sustained circadian expression of the core clock genes Bmal1 (Arnt1), Per2, and Nr1d1 (Rev-erbα) in Nfil3ΔIEC mice. (B) qRT-PCR analysis of epithelial Cd36 and Scd1 expression in germ-free wild-type and conventional Nfil3fl/fl and Nfil3ΔIEC mice at ZT4. (C) Western blot of epithelial CD36 in germ-free (gf) and conventional wild-type (conv) mice, as well as in conventional Nfil3fl/fl and Nfil3ΔIEC mice. All mice were fed a HFD. Mice were sacrificed at ZT4. α-Tubulin was used as the loading control. (D and E) qRT-PCR analysis of epithelial Cd36 and Scd1 expression in conventional wild-type (wt) and ID2-deficient (Id2gfp/gfp) mice (D) and Stat3fl/fl and Stat3ΔIEC mice (E) at ZT4. (F) Oil Red O detection of lipids in the small intestines of Nfil3fl/fl and Nfil3ΔIEC mice fed a HFD. Nuclei were stained with methyl green. Scale bars, 40 μm. (G) Total lipid concentrations in isolated small intestinal epithelial cells (IECs) from Nfil3fl/fl and Nfil3ΔIEC mice fed a HFD. (H) Total neutral lipid concentrations in feces of Nfil3fl/fl and Nfil3ΔIEC mice fed a HFD. Data in (B), (D), (E), (G), and (H) are from N = 5 to 12 mice per group. Means ± SEM (error bars) are plotted; statistics were performed with Student’s t test or one-way ANOVA. *P < 0.05; **P < 0.01.

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  • The intestinal microbiota regulates body composition through NFIL3 and the circadian clock

    Yuhao Wang, Zheng Kuang, Xiaofei Yu, Kelly A. Ruhn, Masato Kubo, Lora V. Hooper

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