Whether and how the gut microbiome affects adipose tissue homeostasis is an area of current investigation. Virtue et al. showed that a high-fat diet in mice led to the activation of miR-181 in white adipose tissue (WAT) and subsequent obesity, insulin resistance, and WAT inflammation. The authors tied the increased expression of this microRNA to a reduction in circulating microbiota-derived metabolites produced by tryptophan metabolism in the gut and confirmed this link by administering indole to mice. miR-181 was increased in WAT and indole was reduced in the plasma of obese humans, suggesting the potential relevance of this axis to human disease.

The gut microbiota is a key environmental determinant of mammalian metabolism. Regulation of white adipose tissue (WAT) by the gut microbiota is a process critical to maintaining metabolic fitness, and gut dysbiosis can contribute to the development of obesity and insulin resistance (IR). However, how the gut microbiota regulates WAT function remains largely unknown. Here, we show that tryptophan-derived metabolites produced by the gut microbiota controlled the expression of the miR-181 family in white adipocytes in mice to regulate energy expenditure and insulin sensitivity. Moreover, dysregulation of the gut microbiota–miR-181 axis was required for the development of obesity, IR, and WAT inflammation in mice. Our results indicate that regulation of miR-181 in WAT by gut microbiota–derived metabolites is a central mechanism by which host metabolism is tuned in response to dietary and environmental changes. As we also found that MIR-181 expression in WAT and the plasma abundance of tryptophan-derived metabolites were dysregulated in a cohort of obese human children, the MIR-181 family may represent a potential therapeutic target to modulate WAT function in the context of obesity.

The miR-181 family of miRNAs is highly conserved in metazoans. Although it has been reported that this miRNA family controls inflammatory pathways contributing to the progression of obesity-associated pathologies ( 12 ), its role in regulating metabolism and inflammation in response to dietary or environmental changes is largely unknown. Herein, we show that specific microbiota-derived metabolites regulate the expression of miR-181 in white adipocytes to tightly control energy expenditure, adiposity, WAT inflammation, and insulin sensitivity. Moreover, we show that dysregulation of the microbiota–miR-181 axis is required for the development of obesity, IR, and WAT inflammation in mice. Our results indicate that the regulation of miRNAs in central metabolic organs by microbiota-derived cues may be a key mechanism by which host metabolism is tuned in response to dietary and environmental changes.

Accumulating evidence indicates that many microRNAs (miRNAs) have evolved to act not only as genetic switches of specific, individual genes but also as control nodes in large gene regulatory networks ( 9 ). Hence, miRNAs can tune large gene expression programs in response to specific cellular and organismal stimuli in a rapid, reversible manner. Several highly conserved miRNAs have been shown to play critical roles in controlling metabolic homeostasis, and their dysregulation contributes to the development of obesity and IR ( 10 , 11 ). However, whether specific miRNAs regulate the function of central metabolic organs in response to microbiota-derived cues is not clear.

The intestinal microbiota produces thousands of small, diffusible metabolites capable of modulating basic physiological processes in the intestine and distant tissues ( 7 ). Some of these metabolites have been shown to regulate metabolic and inflammatory processes involved in the pathogenesis of obesity-associated disorders ( 1 , 8 ). However, the functions of most of these metabolites, and how disturbances in metabolite abundance contribute to obesity and associated pathologies, are poorly understood. Moreover, although it is now clear that WAT mass, function, and inflammation are regulated by the gut microbiota, the role of microbiota-derived metabolites in these processes remains mostly unknown.

The gut microbiota is a key environmental factor that can contribute to the development of obesity, insulin resistance (IR), and associated pathologies ( 1 ). Yet, how the gut microbiota regulates the functions and inflammatory status of central metabolic organs during these disease processes remains poorly understood. White adipose tissue (WAT) is the major site of energy storage in vertebrates and tightly controls glucose homeostasis in mammals ( 1 ). WAT expansion, dysfunction, and inflammation are hallmarks of obesity and play a critical role in the development of highly prevalent metabolic disorders such as IR, atherosclerosis, and nonalcoholic fatty liver disease ( 2 ). In recent years, the gut microbiota has emerged as a central regulator of mammalian adipose tissue. The gut microbiota largely promotes energy storage by decreasing thermogenesis in induced brown adipose tissue (iBAT) and promoting WAT expansion ( 3 ). Moreover, dysbiosis of the gut microbiota during nutritional surplus contributes to the development of obesity and related disorders ( 1 , 2 , 4 ). The microbiota modulates the production and function of iBAT by regulating the inflammatory milieu of this tissue. However, the signals from the microbiota that control WAT functions and inflammation and the molecular mechanisms by which the microbiota regulates this central metabolic organ are largely undetermined ( 1 , 3 – 6 ).

RESULTS

The miR-181 family is elevated in WAT of obese mice and humans The miR-181 family is composed of six highly conserved mature miRNAs transcribed from three independently segregating clusters expressed primarily in hematopoietic cells (Fig. 1A and fig. S1A) (13). To begin interrogating the role of the miR-181 family in metabolic regulation, we measured the expression of each miRNA cluster in different tissues from lean mice fed a normal chow diet (NCD) and obese mice fed a high-fat diet (HFD). All miR-181 clusters were up-regulated in epididymal WAT (eWAT) during diet-induced obesity (DIO), with no changes in expression detected in other tissues examined (Fig. 1, B to D). The MIR-181A2-B2 cluster was likewise strongly up-regulated in adipose tissue from obese adult humans (Fig. 1E), indicating that dysregulation of miR-181 in adipose tissue during DIO is conserved in humans. These results suggest that the miR-181 family may play a critical role in the regulation of WAT and that its dysregulation during DIO could contribute to the pathogenesis of obesity and associated disorders. 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 CO 2 elimination, (K) rate of O 2 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.

The miR-181 family promotes the progression of DIO and fat mass gain Combined ablation of all three miR-181 clusters is embryonic lethal (13). Therefore, to interrogate the role of this miRNA during DIO, we generated mice lacking the two most abundantly expressed miR-181 clusters (miR-181a1-b1−/−; miR-181a2-b2−/−), herein referred to as double-knockout (DKO) mice (13). We then fed wild-type (WT) and DKO mice an NCD or HFD and determined their body composition and change in body weight over time. When fed an NCD, WT and DKO mice did not show differences in body weight, but DKO mice had a mild yet significant (P = 0.008) reduction in total fat mass (Fig. 1, F and G, and fig. S1, B to D). When administered an HFD, miR-181–deficient mice were completely protected from developing obesity concurrent with a substantially lower total fat mass compared to WT mice (Fig. 1, F and G, and fig. S1, B to D). Furthermore, histological examination of eWAT revealed that DKO mice had a reduction in adipocyte size compared to WT mice (Fig. 1H and fig. S1, E to G). Because we did not observe alterations in lean mass (Fig. 1G and fig. S1C), these results indicate that the miR-181 family plays a critical role in promoting fat mass gain, driving the progression of obesity. Differences in body weight and composition are the result of alterations in either caloric intake or energy expenditure (14). To understand how the miR-181 family regulates body weight and composition, we measured whole-body metabolism using a Comprehensive Laboratory Animal Monitoring System (CLAMS) in HFD-fed WT and DKO mice before body weights diverged (fig. S2A). After 6 weeks of HFD feeding, we observed that O 2 consumption (VO 2 ), CO 2 production (VCO 2 ), and calculated heat production were elevated in miR-181–deficient mice (Fig. 1, I to K, and fig. S2, B to D). In addition, DKO mice showed an increase in the respiratory exchange ratio (Fig. 1L and fig. S2E), indicating that miR-181–deficient mice metabolized a mix of carbohydrates and fatty acids for energy. The increased energy expenditure and alterations in substrate utilization in DKO mice were observed without changes in locomotor activity and with increased food intake (fig. S2, F to K). We found similar differences in CLAMS studies performed in the absence of nutritional stress in NCD-fed mice (fig. S3, A to J). We did not observe alterations in nutrient absorption in DKO mice (fig. S3, K and L). Using general guidelines for interpreting mouse metabolic studies (15), these results suggested that decreased fat mass and resistance to DIO in miR-181–deficient mice were the consequence of increased energy expenditure. To determine whether the increased energy expenditure in miR-181–deficient mice was associated with increased beiging of inguinal WAT (iWAT), we performed 6- and 24-hour cold challenges in WT and DKO mice. We observed moderate increases in metabolic and transcriptional indicators of cold-induced thermogenesis in DKO mice compared to WT mice (fig. S4, A to K), consistent with our findings that miR-181 was not induced in iWAT or brown adipose tissue during DIO (fig. S4, L and M). These results suggested that energy expenditure, which can protect against obesity, was increased in miR-181–deficient animals through multiple mechanisms, some of which may be dependent on Ucp1-mediated thermogenesis. Together, these results suggest that aberrantly elevated miR-181 expression in visceral WAT during nutritional surplus in mice and humans is critical for the progression of DIO and associated disorders.

The gut microbiota regulates the expression of the miR-181 family in white adipocytes We then sought to investigate the upstream factors regulating miR-181 in WAT. Cytokines are central regulators of WAT function (39), and it is now well recognized that elevated proinflammatory cytokines promote IR and obesity (40). We hypothesized that cytokines tightly control adipocyte miR-181 expression to regulate WAT inflammation and whole-body glucose and energy homeostasis. To test this hypothesis, we first assessed whether cytokines known to regulate insulin sensitivity and obesity control expression of miR-181 in cultured adipocytes. However, we did not observe changes in miR-181 expression in adipocytes stimulated with pro- or anti-inflammatory cytokines (fig. S7, A to D). Lipopolysaccharide (LPS), a major component of Gram-negative bacterial membranes, potently induces proinflammatory cytokines, IR, and WAT inflammation. To test its effect on adipocyte miR-181 expression in vivo, we administered LPS to mice either orally or by intraperitoneal injection. Consistent with our in vitro data, LPS administration did not affect expression of mature miR-181a or miR-181b in adipocytes in vivo (fig. S7, E and F). These results indicate that neither inflammatory cytokines nor translocation of LPS is the main regulator of miR-181 expression in adipocytes in vivo. It is now well recognized that the gut microbiota is a central regulator of WAT in healthy organisms and promotes WAT dysfunction during DIO (3, 4). Furthermore, microbiota-derived signals have been shown to regulate metabolism, and dysbiosis can promote obesity and associated disorders (1, 41). Therefore, we aimed to establish whether the microbiota regulates miR-181 in adipocytes by comparing miR-181 expression in eWAT from germ-free (GF) and conventionally housed specific pathogen–free (SPF) mice. The presence of microbiota greatly increased expression of mature miR-181a and miR-181b in eWAT adipocytes (Fig. 4, A and B). Expression of miR-181c was primarily increased in eWAT stromal vascular cells, but not adipocytes (fig. S7G), demonstrating that induction of miR-181 family members by the microbiota is cell type specific even within WAT. Our previous results in DKO mice suggested that the miR-181c-d locus was not required for the observed phenotypes (Figs. 1 and 2). To further establish the role of the microbiota in regulating miR-181, we colonized GF mice with microbiota from SPF mice by cohousing and then measured expression of mature miR-181 species in eWAT adipocytes. Colonization of GF mice with the microbiota of SPF mice substantially increased eWAT adipocyte expression of miR-181a and miR-181b (Fig. 4C). Conversely, oral treatment of SPF mice with broad-spectrum antibiotics decreased miR-181 expression in eWAT in vivo (fig. S7H). Furthermore, HFD-induced up-regulation of adipocyte miR-181 expression during obesity was microbiota dependent because miR-181 expression was abrogated in HFD-fed GF and antibiotic-treated mice (Fig. 4, A and B, and fig. S7I). Together, these results indicate that the microbiota regulates expression of the miR-181 family in eWAT adipocytes. Moreover, they suggest that a key mechanism by which the microbiota promotes IR and adiposity is through the regulation of this highly conserved family of miRNAs in eWAT adipocytes. 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.