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Microbiome Remodeling via the Montmorillonite Adsorption-Excretion Axis Prevents Obesity-related Metabolic Disorders - PubMed

Microbiome Remodeling via the Montmorillonite Adsorption-Excretion Axis Prevents Obesity-related Metabolic Disorders

Pengfei Xu et al. EBioMedicine. 2017 Feb.

Abstract

Obesity and its related metabolic disorders are closely correlated with gut dysbiosis. Montmorillonite is a common medicine used to treat diarrhea. We have previously found that dietary lipid adsorbent-montmorillonite (DLA-M) has an unexpected role in preventing obesity. The aim of this study was to further investigate whether DLA-M regulates intestinal absorption and gut microbiota to prevent obesity-related metabolic disorders. Here, we show that DLA-M absorbs free fatty acids (FFA) and endotoxins in vitro and in vivo. Moreover, the combination of fluorescent tracer technique and polarized light microscopy showed that DLA-M crystals immobilized BODIPY® FL C16 and FITC-LPS, respectively, in the digestive tract in situ. HFD-fed mice treated with DLA-M showed mild changes in the composition of the gut microbiota, particularly increases in short-chain fatty acids (SCFA)-producing Blautia bacteria and decreases in endotoxin-producing Desulfovibrio bacteria, these changes were positively correlated with obesity and inflammation. Our results indicated that DLA-M immobilizes FFA and endotoxins in the digestive tract via the adsorption-excretion axis and DLA-M may potentially be used as a prebiotic to prevent intestinal dysbiosis and obesity-associated metabolic disorders in obese individuals.

Keywords: Endotoxins; Free fatty acids; Gut microbiota; Metabolic disorders; Montmorillonite; Obesity.

Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

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Figures

Fig. 1
Fig. 1

DLA-M decreases body weight and improves insulin resistance in HFD C57BL/6J mice. NCD and HFD (60% kcal fat) C57BL/6J mice were treated daily with water or 1 g/kg/day DLA-M by intragastric gavage for 10 weeks (n = 6–15). Effects of DLA-M treatment on the body weight gain and the calculated AUC (a), serum TG and TC content (b), fasting blood glucose and serum insulin levels (c), and calculated HOMA-IR and QUICKI values (d) are shown. IGTT (e); OGTT (f); ITT (g) results. Curves of the blood glucose levels and the calculated AUC are shown. Values are expressed as the means ± s.e.m. **, P < 0.01; ***, P < 0.001 compared with NCD. #, P < 0.05; ##, P < 0.01; and ###, P < 0.001 compared with HFD. Differences were assessed by ANOVA with Tukey's multiple comparison test.

Fig. 2
Fig. 2

DLA-M decreases the fat accumulation in the adipose tissues and livers of HFD mice. Epi-WAT weight and Epi-WAT/BW ratio (a), Per-WAT weight and Per-WAT/BW ratio (b), and Mes-WAT weight and Mes-WAT/BW ratio (c) of the mice described in Fig. 1. (d) H&E staining of Epi-WAT sections from mice described in (a) (scale: 200 μm). Mean epididymal adipocyte size (e) and adipocyte size frequency (f) in each group (n = 5 mice per group). (g) Oil Red O staining of liver sections (scale: 100 μm) and liver TG content (h) of mice. (i) Liver mRNA expression of genes involved in lipogenesis was measured by real-time PCR analysis. (j) The FABP protein production (upper panel) and relative protein level (lower panel) were examined in the liver. Values are shown as the means ± s.e.m. *, P < 0.05; **, P < 0.01; ***, and P < 0.001 compared with NCD. #, P < 0.05; ##, P < 0.01; and ###, P < 0.001 compared with HFD by ANOVA with Tukey's multiple comparison test.

Fig. 3
Fig. 3

DLA-M absorbs FFA and prevents hepatic steatosis. Box-plots of the NEFA concentration in the sera (a) and livers (b) of the mice described in Fig. 1. (c) Under a whole-body imaging scope, the RFP mice were separately treated with DLA-M (1 g/kg) and BODIPY® FL C16 (5 mg/kg) by gavage for 4 h. (d) DLA-M absorbs BODIPY® FL C16 in vitro (the first panels). Gastrointestinal content smears of mice in (c) in different parts (stomach, duodenum, jejunum, ileum, cecum and colon); in the middle panels, FFA appear green, and the lower panels show polarized light microscopy images of DLA-M crystals (scale: 20 μm). (e) BODIPY and Oil Red O staining of steatotic L-02 cells that were treated with a 1 mM free fatty acid (FFA) mixture (oleic acid and palmitic acid at a molar ratio of 2:1) for 48 h after treatment with 0–400 mg/ml DLA-M using a Transwell model (scale: 50 μm). (f) Quantification of the intracellular triglyceride levels in (e). (g) The mRNA expression levels of fatty acid transport and lipogenic genes were measured by real-time PCR. (h) The FABP protein production was examined by western blotting and the relative protein level was normalized to β-actin. Sodium OA (1 mM) that was dissolved in PBS and PA that was dissolved in methanol (1 mM) were treated with 0–400 mg/ml DLA-M: OA content (i) and PA content (j) in the supernatant. (k) Separated intestinal contents of mice fed a NCD or HFD: saline was added (1:20), and the samples were treated with 0–400 mg/ml DLA-M, laumontite and maifanite for 6 h. Then, the relative content of NEFA was measured. Values are shown as the mean ± s.e.m. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 compared with NCD/Con. #, P < 0.05; ##, P < 0.01; and ###, P < 0.001 compared with HFD/FFA by ANOVA with Tukey's multiple comparison test.

Fig. 4
Fig. 4

DLA-M absorbs endotoxin and decreases endotoxemia. Box-plots of the endotoxins (a), TNFα and IL-6 (b) concentrations in the serum of the same mice as in Fig. 1. (c) The liver mRNA expression levels of TNFα, IL-6 and COX-2 were measured by real-time PCR. (d) The liver TLR-4, IκB-α and NF-κB protein production was examined by western blotting and relative protein level was normalized with β-actin. (e) Under a whole-body imaging scope, the RFP mice were separately treated with DLA-M (1 g/kg) and FITC-LPS (5 mg/kg) by gavage for 4 h. (f) DLA-M absorbs FITC-LPS in vitro (the first panels). Gastrointestinal content smears of mice in (e) in different tissues (stomach, duodenum, jejunum, ileum, cecum and colon); in the middle panels, LPS appear green, and the lower panels show polarized light microscopy images of DLA-M crystals (scale: 20 μm). LPS (5 mg/ml) dissolved in PBS was treated with 0–400 mg/ml DLA-M, laumontite and maifanite for 6 h: the LPS content (g) in the supernatant was examined. Values are shown as the mean ± s.e.m. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 compared with NCD. #, P < 0.05; and ##, P < 0.01 compared with HFD by ANOVA with Tukey's multiple comparison test.

Fig. 5
Fig. 5

DLA-M alters the intestinal morphology of HFD-fed mice. (a) Representative images of the gastrointestinal system (stomach, duodenum, jejunum, ileum, cecum and colon). (b) The small intestine length and small intestine/naso-anal length ratio in the mice are notated as in Fig. 1. (c) H&E staining of proximal jejunum sections from mice described in (A) (scale: 100 μm). (d) Proximal jejunum villi length. (e) The intestinal (ileum) mRNA expression of occludin was measured by real-time PCR. Values are shown as the means ± s.e.m. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 compared with NCD. #, P < 0.05; and ##, P < 0.01compared with HFD by ANOVA with Tukey's multiple comparison test.

Fig. 6
Fig. 6

DLA-M alters the gut microbiota composition in HFD mice. (a) OTU-Venn of gut microbiota in the three groups as in Fig. 1. (b) The plots shown were generated using the weighted version of the UniFrac-based PCoA. (c) Bray-Curtis cluster tree using the UPGMA. Comparison of the phylum-level proportional abundance (d), richness represented as the proportions of OTUs classified at the phylum rank (e) (n = 5). *, P < 0.05 and **, P < 0.01 compared with NCD based on the Mann-Whitney test. (f) Heat map showing the relative abundance of RDA-identified key OTUs that were significantly altered by DLA-M in the HFD mice. (g) Representative bacterial taxa information (genus, family and phylum) of 77 OTUs from (f) are shown. White circles and black triangles indicate the OTUs that increased or decreased, respectively, in the NCD and HFD + DLA-M groups relative to the HFD group on the basis of the Turkey (HSD) test (P < 0.05).

Fig. 7
Fig. 7

LEfSe identifies the most differently abundant taxons in the gut microbiota. (a) Only taxa meeting a significant LDA threshold of > 4 are shown in the feces of mice as notated in Fig. 1. The relative abundance of Blautia (b) and Desulfovibrio (c) was obtained from the fecal microbiota from LEfSe results. Solid and dashed lines indicate the mean and median, respectively.

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