Ganoderma lucidum reduces obesity in mice by modulating the composition of the gut microbiota

Abstract

Obesity is associated with low-grade chronic inflammation and intestinal dysbiosis. Ganoderma lucidum is a medicinal mushroom used in traditional Chinese medicine with putative anti-diabetic effects. Here, we show that a water extract of Ganoderma lucidum mycelium (WEGL) reduces body weight, inflammation and insulin resistance in mice fed a high-fat diet (HFD). Our data indicate that WEGL not only reverses HFD-induced gut dysbiosis-as indicated by the decreased Firmicutes-to-Bacteroidetes ratios and endotoxin-bearing Proteobacteria levels-but also maintains intestinal barrier integrity and reduces metabolic endotoxemia. The anti-obesity and microbiota-modulating effects are transmissible via horizontal faeces transfer from WEGL-treated mice to HFD-fed mice. We further show that high molecular weight polysaccharides (>300 kDa) isolated from the WEGL extract produce similar anti-obesity and microbiota-modulating effects. Our results indicate that G. lucidum and its high molecular weight polysaccharides may be used as prebiotic agents to prevent gut dysbiosis and obesity-related metabolic disorders in obese individuals.

Figure 1. WEGL reduces body weight and fat accumulation in HFD-fed mice.

Chow- and HFD-fed mice were treated daily with 100 μl of either water or WEGL at 2, 4 or 8% (w/v) by intragastric gavage for two months (n=7 for each group). Effects of WEGL treatment on body weight (a) body weight gain (b) epididymal fat (c) subcutaneous fat (d) and epididymal adipocyte size (e) are shown. In e, adipocyte size was estimated using the Image J software (lower panel). Scale bar, 50 μm. Liver weight was measured in HFD and control, chow-fed mice (f). Liver lipid content was assessed using oil red O staining (g). Scale bar, 30 μm. Data are expressed as mean±s.e.m. Body weight differences in a were analysed using unpaired two-tailed Student’s t-test (**P<0.01, ***P<0.001). Graph bars in b, c, d and f marked with different letters on top represent statistically significant results (P<0.05) based on Newman–Keuls post hoc one-way ANOVA analysis, whereas bars labelled with the same letter correspond to results that show no statistically significant differences. In the case where two letters are present on top of the bar in b, each letter should be compared separately with the letters of other bars to determine whether the results show statistically significant differences.

Figure 2. WEGL decreases pro-inflammatory cytokine expression in the liver and adipose tissues of HFD-fed mice.

Animals were treated as in Fig. 1. Relative expression of TNF-α (a), IL-1β (b), IL-6 (c), IL-10 (d) and PAI-1 (e) in hepatic and adipose tissues was assessed using qRT–PCR and in comparison with the Chow group. Data are shown as mean±s.e.m. Graph bars with different letters on top represent statistically significant results (P<0.05) based on Newman–Keuls post hoc one-way ANOVA analysis, whereas bars with the same letter correspond to results that show no statistically significant differences. In the case where two letters are present on top of the bars in d,e, each letter should be compared separately with the letters of other bars to determine whether the results show statistically significant differences.

Figure 3. WEGL reduces serum LPS and TLR4-related signalling pathways in HFD mice.

Effects of WEGL treatment on serum endotoxin (a) TLR4 protein production (b,c) JNK phosphorylation (d,e) and IκB-α production (f,g) were examined in the liver and epididymal adipose tissues of chow- and HFD-fed mice as described in Fig. 1. Serum endotoxin (EU ml⁻¹) was determined as mean±s.e.m. using the limulus amebocyte lysate assay kit. Representative immunoblots for target proteins in b-g are shown. Molecular weight markers were indicated as kilodaltons (kDa). Protein levels were normalized to internal controls (β-actin or total JNK, T-JNK) and the relative ratio to the Chow group was labelled on the top of immunoblots. Graph bars in a with different letters on top represent statistically significant results (P<0.05) based on Newman–Keuls post hoc one-way ANOVA analysis, whereas bars labelled with the same letter correspond to results that show no statistically significant differences. Where two letters are present on top of the bar, each letter should be compared separately with the letters of other bars to determine whether the results show statistically significant differences.

Figure 4. WEGL alters microbiota composition in HFD-fed mice.

Microbiota composition in faeces of chow-fed mice treated with or without 8% WEGL and HFD mice treated with 2, 4 or 8% WEGL were analysed using next generation sequencing (n=7 for each group). (a) Plots shown were generated using the weighted version of the UniFrac-based PCoA. (b) Multivariate analysis of variance from PCoA matrix scores. (c) Bacterial taxonomic profiling in the phylum level of intestinal bacteria from different mouse groups. (d) Heatmap showing the abundance of 91 OTUs significantly altered by WEGL in HFD-fed mice based on RDA. (e) Represented bacterial taxa information (species, genus, family and phylum) of 91 OTUs from d are shown. White circles and black diamonds indicate the OTUs that increased or decreased in Chow- and HFD+WEGL-fed groups relative to the HFD-fed group. Black stars represent OTUs whose abundance in chow-fed mice was altered by HFD and then reversed by WEGL. OTU taxonomy is shown on the right.

Figure 5. Obesity and fat accumulation are reversed by faecal transplantation from WEGL-treated mice to HFD-fed mice.

Eight-week-old HFD-fed mice were colonized with faeces from different mouse groups for 8 weeks, followed by measurement of body weight (a) epididymal fat (b) subcutaneous fat (c) and liver weight (d). Each group consisted of five mice. Body weight differences in a were analysed using unpaired two-tailed Student’s t-test (*P<0.05, **P<0.01, ***P<0.001). Graph bars in b–d with different letters on top represent statistically significant results (P<0.05) based on Newman–Keuls post hoc one-way ANOVA analysis, whereas data labelled with the same letter correspond to results that show no statistically significant differences.

Figure 6. Analysis of pro-inflammatory cytokines and intestinal tight junctions following faecal transplantation from WEGL-treated mice.

Eight-week-old HFD mice were colonized with faeces from the indicated mouse groups for 8 weeks. In comparison with the HFD→HFD group, relative mRNA expression levels of TNF-α (a) IL-1β (b) IL-6 (c) and MCP-1 (d) in hepatic and adipose tissues as well as occludin (e) and ZO-1 (f) in ileum, were assessed using qRT–PCR. Representative ileum immunoblots for occludin, ZO-1 and β-actin in each group. (g) Molecular weight markers were indicated as kDa. Each group consisted of five mice. Graph bars in a–f with different letters on top represent statistically significant results (P<0.05) based on Newman–Keuls post hoc one-way ANOVA analysis, whereas bars labelled with the same letter correspond to results with no statistically significant differences.

Figure 7. Analysis of gut microbiota following faecal transplantation.

Faecal transplantation from Chow-, HFD- and Chow/HFD+8% WEGL-fed mice was performed and relevant microbiota analysis was done as described in the Methods. (a) The plots shown were generated using the weighted version of UniFrac-based PCoA. (b) Multivariate analysis of variance from PCoA matrix scores. (c) Bacterial taxonomic analysis of intestinal bacterium from each mouse groups. (d) Heatmap showing the abundance of 155 OTUs significantly altered by Chow, Chow+8% WEGL and HFD+8% WEGL transplanted mice based on RDA analysis. (e) Represented bacterial taxa information (species, genus, family and phylum) of 155 OTUs from d are shown. White circles and black diamonds indicate the OTUs that increased or decreased compared with HFD recipient mice.

Figure 8. Effect of WEGL polysaccharide fractions on body weight and fat accumulation in HFD-fed mice.

Mice fed with chow or HFD were treated daily with 100 μl of polysaccharide subfractions (G1, G2, G3, G4), 8% WEGL or water by intragastric gavage for 2 months (n=5 for each group). Effects of polysaccharide subfractions on body weight (a) body weight gain (b) epididymal fat (c) subcutaneous fat (d) and liver weight (e). Body weight differences in a were analysed using unpaired two-tailed Student’s t-test (**P<0.01, ***P<0.001). Graph bars in b–e labelled with different letters on top represent statistically significant results (P<0.05) based on Newman–Keuls post hoc one-way ANOVA analysis, whereas bars with the same letter correspond to results that show no statistically significant differences. In the case where two letters are present on top of the bars in e, each letter should be compared separately with the letters of the other bars to determine whether the results show statistically significant differences.