Beneficial Effects of Newly Isolated Akkermansia muciniphila Strains from the Human Gut on Obesity and Metabolic Dysregulation

Abstract

The identification of new probiotics with anti-obesity properties has attracted considerable interest. In the present study, the anti-obesity activities of Akkermansia muciniphila (A. muciniphila) strains isolated from human stool samples and their relationship with the gut microbiota were evaluated using a high fat-diet (HFD)-fed mice model. Three strains of A. muciniphila were chosen from 27 isolates selected based on their anti-lipogenic activity in 3T3-L1 cells. The anti-lipogenic, anti-adipogenic and anti-obesity properties of these three strains were evaluated further in HFD-induced obese mice. The animals were administered these strains six times per week for 12 weeks. The treatment improved the HFD-induced metabolic disorders in mice in terms of the prevention of body weight gain, caloric intake and reduction in the weights of the major adipose tissues and total fat. In addition, it improved glucose homeostasis and insulin sensitivity. These effects were also associated with the inhibition of low-grade intestinal inflammation and restoration of damaged gut integrity, prevention of liver steatosis and improvement of hepatic function. These results revealed a difference in the distribution pattern of the gut microbial communities between groups. Therefore, the gut microbial population modulation, at least in part, might contribute to the beneficial impact of the selected A. muciniphila strains against metabolic disorders.

Keywords: Akkermansia muciniphila; anti-obesity; gut microbiota; metabolic disorders; mice; probiotics.

Figure 1

Effects of Akkermansia muciniphila strains on lipid accumulation in 3T3-L1 adipocytes. (a) After differentiation, the intracellular lipid droplets of adipocytes in different experimental groups were visualized under a microscope at 200× magnification upon staining of the cells with Oil Red O. (b) Intracellular deposition of the lipids was measured after extraction and reading the absorbance at 500 nm. The data are presented as the mean ± SEM. *** p < 0.001 versus the control group.

Figure 2

Effects of A. muciniphila treatment on the vital obesity parameters in high fat-diet (HFD)-fed mice. (a) Bodyweight measured weekly, (b) total body weight gain, (c) caloric intake, (d) weight of total fat (subcutaneous fat + epididymal fat + mesenteric fat), (e) serum level of total cholesterol (TC) and (f) serum triglyceride (TG) level in different experimental groups. The data are presented as the mean ± SEM (n = 9–12). Statistical analyses were performed by one-way ANOVA or a Student’s t-test. * p < 0.05, ** p < 0.01 and *** p < 0.001 versus the HFD group. ^# p < 0.05.

Figure 3

A. muciniphila strains improved insulin sensitivity and glucose homeostasis in HFD-induced obese mice. (a) Serum glucose levels at different time-points in oral glucose tolerance tests (OGTT). (b) Serum insulin level and (c) HOMA-IR. mRNA levels of glucose homeostasis (d) G6Pase and (e) GLUT2 in the hepatic of each group (n = 9–12 mice/group). mRNA expression of related markers of gut hormones (f) GLP-1 and (g) PYY were measured in the intestinal tissue (n = 9–12 mice/group). The data are represented as the mean ± SEM. Statistics were performed with one-way ANOVA or Student’s t-test. * p < 0.05, ** p < 0.01 and *** p < 0.001 versus the HFD group.

Figure 4

A. muciniphila administration alleviated hepatic steatosis and improved hepatic function in mice. (a) Histological analysis of H&E stain on the liver sections of mice (n = 3 per condition, scale bar, 100 μm). (b) Representative Oil Red O staining for fat deposition measurement in the liver. (c) The liver steatosis area. Liver steatosis characterized by micro- and macrovacuolization. (d) Oil Red O stained fat deposition area. (n = 3 per condition, scale bar, 50 μm). Representative mRNA expression of (e) PPARγ, (f) SREBP1c, (g) FAS and (h) ACC1 in the liver tissue from mice (n = 9–12 mice/group). (i) The protein expression of the liver tissue (n = 4 mice/group). The data are represented as the mean ± SEM. Statistics were performed with one-way ANOVA or a Student’s t-test. * p < 0.05, ** p < 0.01 and *** p < 0.001 versus the HFD group. ^# p < 0.05.

Figure 5

Effects of A. muciniphila on the adipokine profile of adipose tissue in HFD-fed mice. (a) Histological analysis (H&E staining) on sections of mesenteric fat tissues (n = 3 per condition, scale bar, 100 μm). (b) Average diameters of adipocytes in randomly chosen fields were measured and presented as pixels using Image-Pro Plus 6.0. (c) LPL, (d) FAS, (e) SREBP1c, (f) CD36, (g) IRS1 and (h) Leptin mRNA levels in the adipose tissues (n = 9–12). Data are represented as the mean ± SEM. Statistics were performed with one-way ANOVA or Student’s t-test. * p < 0.05, ** p < 0.01 and *** p < 0.01 versus the HFD group. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001.

Figure 6

A. muciniphila treatment had anti-inflammatory effects on the colon of the HFD-fed mice. mRNA levels of inflammatory cytokines (a) TNF-α, (b) IL-6, (c) IL-10, (d) MCP-1, (e) TLR2 and (f) TLR4 in the intestine tissues of each group. The data are represented as the mean ± SEM. The data were analyzed with one-way ANOVA or Student’s t-test. * p < 0.05, ** p < 0.01 and *** p < 0.001 versus the HFD group. ^# p < 0.05.

Figure 7

A. muciniphila administration improved the intestinal structure and barrier integrity of the HFD-fed mice. (a) Representative microscopic images demonstrating Alcian blue (AB)-staining of the colonic tissue sections of mice from different experimental groups at a magnification of 200×, (b) proportion of AB-positive area (%), (c) number of goblet cells and (d) length of a villus in colonic tissue sections (n = 3 per condition, scale bar, 100 μm). Representative mRNA expression of (e) Muc2, (f) ZO-1 and (g) Occludin in the intestine tissue from mice (n = 9–12 mice/group). (h) The protein expression of the intestine tissue (n = 4 mice/group). Data are represented as the mean ± SEM. Statistics were performed with one-way ANOVA or Student’s t-test. * p < 0.05, ** p < 0.01 and *** p < 0.001 versus the HFD group. ^## p < 0.01.

Figure 8

Effects of A. muciniphila strains on the gut microbiota composition. The gut microbiota was determined by 16S rRNA gene analysis of fecal samples from the mice. (a–c) Principal component analysis (PCA) of the gut microbiota metagenomic samples. (d) Cladogram generated by LEfSe analysis. The LEfSe plot shows enriched bacteria in all phenotypic categories. (e–g) LEfSe was used to identify the bacteria differentially represented between EB-AMDK 10 and HFD (e), EB-AMDK 19 and HFD (f) and EB-AMDK 27 and HFD (g). Only the taxa meeting a linear discriminant analysis (LDA) score threshold of two are listed.