Research ArticleObesity

The gut microbiota regulates white adipose tissue inflammation and obesity via a family of microRNAs

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Science Translational Medicine  12 Jun 2019:
Vol. 11, Issue 496, eaav1892
DOI: 10.1126/scitranslmed.aav1892
  • Fig. 1 The miR-181 family is a critical regulator of adipose tissue function.

    (A) Genomic location of the three miR-181 clusters (miR-181a1-b1, miR-181a2-b2, and miR-181c-d) in mice. (B to D) Expression of pri-miR-181 clusters relative to Hprt in tissue from mice fed an NCD or an HFD, shown as fold change relative to NCD-fed values (n = 3 per group, two independent experiments). (E) Human pri-MIR-181A2-B2 expression in lean subcutaneous [body mass index (BMI) < 24; n = 12], obese subcutaneous (BMI > 30; n = 7), and obese visceral WAT (BMI > 30; n = 7). Each dot represents a single donor. (F) Body weights of WT and miR-181a1-b1−/−; miR-181a2-b2−/− (DKO) mice fed an NCD or an HFD from 6 to 18 weeks of age (WT NCD, n = 10; DKO NCD, n = 5; WT HFD, n = 10; DKO HFD, n = 8). (G) Body composition by magnetic resonance imaging of WT and DKO mice fed an NCD or an HFD from 6 to 18 weeks of age [one dot per mouse (WT NCD, n = 10; DKO NCD, n = 5; WT HFD, n = 10; DKO HFD, n = 8)]. n.s., not significant. (H) Representative hematoxylin and eosin (H&E) staining images of eWAT from WT and DKO mice after 12 weeks of HFD. Scale bars, 50 μm. Quantification of individual adipocytes within a 20× field [one dot per cell (WT NCD, n = 5; DKO NCD, n = 4; WT HFD, n = 5; DKO HFD, n = 3)]. (I to L) Evaluation of whole-body metabolism by CLAMS of WT (n = 5) and DKO (n = 4) mice fed an HFD for 6 weeks starting at 8 to 10 weeks of age. (I) Average calculated heat production, (J) rate of CO2 elimination, (K) rate of O2 consumption, and (L) calculated respiratory exchange ratio (RER). One dot per mouse. Error bars indicate means ± SEM. Two-tailed Student’s t test (B, D, I, J, K, and L), one-way analysis of variance (ANOVA) (E), two-way ANOVA (F), or Mann-Whitney test (C, G, and H). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 2 The miR-181 family regulates insulin sensitivity and promotes a proinflammatory status in WAT during obesity.

    (A) Glucose tolerance test (GTT) of WT and miR-181a1-b1−/−; miR-181a2-b2−/− (DKO) mice fed an NCD. (B) ITT of WT and DKO mice fed an NCD. (C) GTT area under the curve (AUC) values for WT (n = 10) and DKO (n = 5) NCD mice. (D) ITT AUC values for WT (n = 10) and DKO (n = 4) NCD mice. (E) GTT of WT and DKO mice fed an HFD from 6 to 18 weeks of age. (F) ITT of WT and DKO mice fed an HFD at 6 to 18 weeks of age. (G) GTT AUC values for WT (n = 10) and DKO (n = 8) HFD mice. (H) ITT AUC values for WT (n = 9) and DKO (n = 7) HFD mice. One dot per mouse. (I) Serum insulin and (J) HOMA-IR index values for WT and DKO mice fed an NCD or an HFD for 12 weeks [one dot per mouse (WT NCD, n = 8; DKO NCD, n = 7; WT HFD, n = 9; DKO HFD, n = 7)]. (K) Representative Western blot for p-AKT, AKT, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in eWAT of WT (n = 5) and DKO (n = 3) mice fed an HFD from 6 to 18 weeks of age. Mice were fasted for 4 hours before intraperitoneal insulin injection, and after 15 min, eWAT was snap-frozen. p-AKT expression is shown normalized by total AKT and GAPDH. One dot per mouse per lane. a.u., arbitrary units. (L) Representative flow cytometry plots of ILC2s, eosinophils, and Tregs isolated from eWAT of WT and DKO mice fed an HFD from 6 to 18 weeks of age. (M) Quantification of (L) total lymphocytes per gram of adipose tissue and percentage of indicated cell types within the CD45+ immune cell population [one dot per mouse (WT HFD, n = 5 to 10; DKO HFD, n = 3 to 6)]. (N) Expression of M1 and M2 genes in eWAT of HFD-fed mice (WT, n = 5; DKO, n = 4) normalized to Hprt and shown as fold change relative to WT. Error bars show means ± SEM. Two-tailed Student’s t test (D, G, H, K, and N), two-way ANOVA (A, B, E, and F), or Mann-Whitney test (C, I, J, and M). *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 3 Adipocyte-specific expression of the miR-181 family affects glucose homeostasis and body weight.

    (A and B) RNA sequencing (RNA-seq) analysis of eWAT from HFD-fed WT and DKO mice. (A) Gene set enrichment analysis (GSEA) of predicted miR-181 targets [normalized enrichment score (NES), −1.11; enrichment score (ES), −0.24; FDR < 0.32], with heat map of the top predicted targets that were significantly differentially expressed (FDR < 0.05) in WT (n = 4) or DKO (n = 4) HFD mice. Orange highlights indicate genes implicated in adipose tissue development and function. Pink highlights indicate genes implicated in regulation of or response to insulin signaling. (B) GSEA of inflammatory (NES, 1.54; ES, −0.57; FDR < 0.26) and oxidative phosphorylation (NES, −1.09; ES, −0.33; FDR < 0.71) gene sets. (C to G) GTT of TcKO mice fed an HFD from 6 to 18 weeks of age and expressing Cre-recombinase under the control of the following promoters: (C) Cluster of differentiation 4 (CD4) promoter (T cells; Cre n = 8, Cre+ n = 19, five independent experiments), (D) Forkhead box P3 (FoxP3) promoter (Tregs; Cre n = 5, Cre+ n = 12, four independent experiments), (E) Lysozyme 2 (LyzM) promoter (macrophages; Cre n = 8, Cre+ n = 8, two independent experiments), (F) Albumin promoter (hepatocytes; Cre n = 8, Cre+ n = 8), or (G) Fatty acid binding protein 4 (Fabp4) promoter (adipocytes; Cre n = 11, Cre+ n = 10, two independent experiments). (H) ITT of Fabp4 TcKO mice fed an HFD from 6 to 18 weeks of age (Fabp4; Cre n = 24, Cre+ n = 29, three independent experiments. (I) AUC of data from (G) and (H). (J) Body weights of Fabp4 TcKO mice fed an HFD from 6 to 18 weeks of age (Fabp4; Cre n = 11, Cre+ n = 17, four independent experiments). (K) Quantification of adipocyte-specific insulin sensitivity via suppression of FFA production during fasting-refeeding. NCD-fed WT (n = 5) and DKO mice (n = 4 to 5) were fasted for 18 hours and then given NCD ad libitum for 4 hours. Serum FFAs were measured after fasting and refeeding periods. One dot per mouse. Error bars indicate means ± SEM. Two-tailed Student’s t test (I and K) or two-way ANOVA (C to H and J). *P < 0.05, ****P < 0.0001.

  • Fig. 4 Microbiota-derived metabolites regulate the miR-181 family in white adipocytes to control progression to obesity.

    (A and B) Expression of mature (A) miR-181a or (B) miR-181b from adipocyte and stromal vascular (SV) fractions of eWAT from SPF mice fed an NCD and GF mice fed an NCD or an HFD. Expression was normalized to U6 and is shown as fold change relative to NCD GF (NCD SPF, n = 8; NCD GF, n = 6; HFD GF, n = 6; two independent experiments). (C) Expression of mature miR-181a or miR-181b from the adipocyte fraction of eWAT of GF mice (n = 6) and cohoused GF (n = 6) and SPF (n = 6) mice for 8 weeks. Expression was normalized to U6 and is shown as fold change relative to GF. (D) Volcano plot of metabolite abundance in the cecal contents of NCD- or HFD-fed mice, plotted as relative abundance in HFD compared to NCD mice. Metabolites related to tryptophan metabolism are plotted in red. (E) Heat map of the 10 metabolites from (D) most significantly altered in HFD-fed mice compared to NCD-fed mice. (F) Schematic of the conversion of tryptophan to indole, I3CA, and indoxyl sulfate showing the role of tryptophanase. (G) Concentrations of indoxyl sulfate determined by mass spectrometry in plasma from 7- to 9-week-old SPF and GF mice fed an NCD or an HFD for 5 weeks. (H) Abundance of plasma indole determined by mass spectrometry from children stratified by obesity status. Using BMI percentiles for age and gender, individuals were binned into healthy weight (<85th percentile, n = 19), class I obesity (100 to 120% of 95th percentile, n = 10), class II obesity (120 to 140% of 95th percentile, n = 6), and class III obesity (>140% of 95th percentile, n = 3). (I) Lipid accumulation in cultured adipocytes after 6-day treatment with tryptophan-derived metabolites (200 μM), plotted as percentage change in Oil Red O staining in metabolite-treated relative to dimethyl sulfoxide (DMSO)–treated cells. Four independent experiments, three to six biological replicates per group. Trp, tryptophan; N-A-Trp, N-acetyl-tryptophan; ILA, indole lactic acid; IAA, indole acetic acid; 5-HT, 5-hydroxytryptamine (serotonin); KYN, kynurenine; KYNA, kynurenic acid; XA, xanthurenate; IS, indoxyl sulfate. (J) Representative image of cultured adipocytes treated with DMSO or indole (200 μM) and stained with Oil Red O. Scale bars, 2.5 mM. (K) Expression of mature miR-181a and miR-181b in differentiated 3T3-L1 cells after 2-day treatment with indole (100 μM). (L) Body weight and (M) GTT of HFD-fed mice intraperitoneally injected with indole (50 mg/kg; n = 4 to 5) or solvent control (n = 7). Weights measured three times per week and pooled. (N and O) Expression of mature (N) miR-181a or (O) miR-181b from adipocyte fractions of eWAT from mice in (L) and (M), normalized to U6 and shown as fold change relative to solvent-injected mice (one dot per mouse). (P) GTT of miR-181 TcKO Fabp4 HFD-fed mice intraperitoneally injected with indole (50 mg/kg; n = 3) or solvent control (n = 3) three times per week for 7 weeks. (Q and R) Expression of mature miR-181a (Q) or miR-181b (R) from adipocyte fractions of eWAT of SPF mice maintained fed a tryptophan-sufficient (Trp-suff., n = 10) or tryptophan-deficient (Trp-def., n = 10) diet. Expression was normalized to U6 and is shown as fold change relative to Trp-suff. diet; two independent experiments. (S) Body weight and (T) GTT of mice colonized with a WT parental E. coli strain or tnaA E. coli knockout (tnaA KO) (n = 4 per group). Error bars show means ± SEM. Two-tailed Student’s t test (G, I, N, O, and R), one-way ANOVA (A, B, C, and H), two-way ANOVA (L, M, P, S, and T), or Mann-Whitney test (K and Q). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/496/eaav1892/DC1

    Materials and Methods

    Fig. S1. Expression of the miR-181 family affects adipocyte function and body composition.

    Fig. S2. miR-181–deficient mice have increased energy expenditure during HFD feeding.

    Fig. S3. miR-181–deficient mice have increased energy expenditure at baseline.

    Fig. S4. miR-181–deficient mice have increased energy expenditure in the context of a cold challenge.

    Fig. S5. Expression of miR-181 regulates glucose homeostasis and WAT inflammatory status during obesity.

    Fig. S6. Conditional deletion of the miR-181 family in T cells, Tregs, macrophages, or hepatocytes does not affect glucose homeostasis or body composition.

    Fig. S7. Inflammatory cytokines and LPS are not regulators of miR-181 expression.

    Fig. S8. Indole and its hepatic metabolic by-product, indoxyl sulfate, inhibit the expression of miR-181 and adipocyte function.

    Fig. S9. Working model: The gut microbiota regulates progression of obesity and WAT inflammation through a miRNA family expressed in white adipocytes.

    Data file S1. Raw data from figures.

    References (4650)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Expression of the miR-181 family affects adipocyte function and body composition.
    • Fig. S2. miR-181–deficient mice have increased energy expenditure during HFD feeding.
    • Fig. S3. miR-181–deficient mice have increased energy expenditure at baseline.
    • Fig. S4. miR-181–deficient mice have increased energy expenditure in the context of a cold challenge.
    • Fig. S5. Expression of miR-181 regulates glucose homeostasis and WAT inflammatory status during obesity.
    • Fig. S6. Conditional deletion of the miR-181 family in T cells, Tregs, macrophages, or hepatocytes does not affect glucose homeostasis or body composition.
    • Fig. S7. Inflammatory cytokines and LPS are not regulators of miR-181 expression.
    • Fig. S8. Indole and its hepatic metabolic by-product, indoxyl sulfate, inhibit the expression of miR-181 and adipocyte function.
    • Fig. S9. Working model: The gut microbiota regulates progression of obesity and WAT inflammation through a miRNA family expressed in white adipocytes.
    • References (4650)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Raw data from figures.

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