Supplementation with Akkermansia muciniphila in overweight and obese human volunteers: a proof-of-concept exploratory study

Abstract

Metabolic syndrome is characterized by a constellation of comorbidities that predispose individuals to an increased risk of developing cardiovascular pathologies as well as type 2 diabetes mellitus1. The gut microbiota is a new key contributor involved in the onset of obesity-related disorders2. In humans, studies have provided evidence for a negative correlation between Akkermansia muciniphila abundance and overweight, obesity, untreated type 2 diabetes mellitus or hypertension3-8. Since the administration of A. muciniphila has never been investigated in humans, we conducted a randomized, double-blind, placebo-controlled pilot study in overweight/obese insulin-resistant volunteers; 40 were enrolled and 32 completed the trial. The primary end points were safety, tolerability and metabolic parameters (that is, insulin resistance, circulating lipids, visceral adiposity and body mass). Secondary outcomes were gut barrier function (that is, plasma lipopolysaccharides) and gut microbiota composition. In this single-center study, we demonstrated that daily oral supplementation of 1010 A. muciniphila bacteria either live or pasteurized for three months was safe and well tolerated. Compared to placebo, pasteurized A. muciniphila improved insulin sensitivity (+28.62 ± 7.02%, P = 0.002), and reduced insulinemia (-34.08 ± 7.12%, P = 0.006) and plasma total cholesterol (-8.68 ± 2.38%, P = 0.02). Pasteurized A. muciniphila supplementation slightly decreased body weight (-2.27 ± 0.92 kg, P = 0.091) compared to the placebo group, and fat mass (-1.37 ± 0.82 kg, P = 0.092) and hip circumference (-2.63 ± 1.14 cm, P = 0.091) compared to baseline. After three months of supplementation, A. muciniphila reduced the levels of the relevant blood markers for liver dysfunction and inflammation while the overall gut microbiome structure was unaffected. In conclusion, this proof-of-concept study (clinical trial no. NCT02637115 ) shows that the intervention was safe and well tolerated and that supplementation with A. muciniphila improves several metabolic parameters.

Conflict of interest statement

Competing financial interests

A.E., C.D., H.P., P.D.C. and W.M.d.V. are inventors of patent applications (PCT/EP2013/073972; PCT/EP2016/071327, PCT/EP2016/060033 filed in European Patent Office (EP), Australia (AU), Brazil (BR), Canada (CA), China (CN), Eurasian Patent Organization (EA), Israel (IL), India (IN), Hong Kong (HK), Japan (JP), South Korea (KR), Mexico (MX), New Zealand (NZ), and the United States (US)) dealing with the use of A.muciniphila and its components in the context of obesity and related disorders. P.D.C. and W.M.d.V. are co-founders of A-Mansia Biotech SA.

Figures

Extended data figure 1. Flow chart of the interventional study.

Diagram of the participant selection procedure, with the following information: number of individuals enrolled at each step of the study progress; number of individuals included in the final analysis; details of events that led to a reduction in the group size.

Extended data figure 2. Changes in inflammatory parameters and GLP-1.

(a) Soluble CD40 Ligand (sCD40L), (b) Growth-related oncogene (GRO/CXCL1), (c) Monocyte Chemoattractant Protein-1 (MCP-1) and (d) Glucagon-like peptide-1 (GLP-1). Differential values (MD and MD from placebo) are expressed as mean ± SEM, either as raw data or as percentages. Bars represent mean change from baseline value per group, with their standard errors. Mann-Whitney tests or unpaired T-tests were performed to compare differential values of both treated groups versus the Placebo group (inter-group changes), according to the distribution. The respective P value are indicated in the table. Lines represent raw values before and after 3 months of supplementation. Distribution of values within each group for each timing is illustrated by a box and whisker. In the box plots, the line in the middle of the box is plotted at the median, the inferior and superior limit of the box correspond to the 25th and the 75th percentiles respectively. Wilcoxon matched-pairs signed ranks tests or Paired T-tests were performed to verify changes from baseline (intra-group changes), according to the distribution and, when drawn, the capped line above the concerned group shows the corresponding P-value. Changes between 0 and 3 months across the 3 groups were analyzed with Kruskal-Wallis or One-way ANOVA according to the distribution and group-wise comparisons were performed using Bonferroni and Tuckey’s adjustment for multiple testing, respectively. MD, Mean Difference. Placebo, n = 11; Pasteurized, n =12; Alive, n=9 for all parameters except for GRO: Placebo, n = 7; Pasteurized, n =10; Alive, n=8. All tests were performed in two-tailed.

Extended data figure 3. Changes in faecal microbiome.

(a)Akkermansia muciniphila abundance in feces evaluated by Q-PCR. Differential values (MD and MD from placebo) are expressed as mean ± SEM as raw. Bars represent mean change from baseline value per group, with their standard errors. Mann-Whitney tests were performed to compare differential values of both treated groups versus the Placebo group (inter-group changes), according to the distribution. The respective P value are indicated in the table. Lines represent raw values before and after 3 months of supplementation. Distribution of values within each group for each timing is illustrated by a box and whisker. In the box plots, the line in the middle of the box is plotted at the median, the inferior and superior limit of the box correspond to the 25th and the 75th percentiles respectively. Wilcoxon matched-pairs signed ranks tests were performed to verify changes from baseline (intra-group changes), according to the distribution. When the difference is significative, a capped line is marked above the concerned group with the corresponding P value. Kruskal-Wallis analysis were used to compare changes between 0 and 3 months across the 3 groups, according to the distribution. MD, Mean Difference. Placebo, n = 11; Pasteurized, n =12; Alive, n=9. All tests were performed in two-tailed. *P < 0.05. (b) Visualization of the participants’ faecal microbiota composition at baseline and endpoint of the intervention. Faecal microbiota dissimilarity between samples is represented by principal coordinates analysis (genus-level Aitchison distance), with 6 sample groups - corresponding to the 3 different treatment arms at baseline or at endpoint represented by confidence ellipses (80% confidence interval). Intervention effects are symbolized by colored arrows, with direction and length corresponding to the shift in group centroid coordinates from baseline to endpoint for each treatment arm (re-scaled x4 and re-centered at baseline global centroid). Placebo, n = 11; Pasteurized, n =12; Alive, n=9.

Figure 1. Changes in parameters related to glucose metabolism and WBC.

(a) Insulinemia, (b) Glycemia, (c) Insulin Resistance score, (d) Insulin Sensitivity, (e) DDPIV activity (f) and With blood cell count. Differential values (MD and MD from placebo) are expressed as mean + SEM, either as raw data or as percentages. Bars represent mean change from baseline value per group, with their standard errors. Mann-Whitney tests or unpaired T-tests were performed to compare differential values of both treated groups versus the Placebo group (inter-group changes), according to the distribution. The respective P value are indicated in the table and when the test is significative, bars are marked by * symbol. Lines represent raw values before and after 3 months of supplementation. Distribution of values within each group for each timing is illustrated by a box and whisker. In the box plots, the line in the middle of the box is plotted at the median, the inferior and superior limit of the box correspond to the 25th and the 75th percentiles respectively. The whiskers correspond to the minimum and maximum value. Wilcoxon matched-pairs signed ranks tests or Paired T-tests were performed to verify changes from baseline (intra-group changes), according to the distribution. When the difference is significative, a capped line is marked above the concerned group with the corresponding P value. Changes between 0 and 3 months across the 3 groups were analyzed with Kruskal-Wallis or One-way ANOVA according to the distribution and group-wise comparisons were performed using Bonferroni and Tuckey’s adjustment for multiple testing, respectively. When the difference is significative, a line is marked above the concerned groups with the corresponding P value. DPPIV, dipeptidyl peptidase-IV ; MD, Mean Difference; WBC, white blood cells. Placebo, n = 11; Pasteurized, n =12; Alive, n=9 for all parameters except for WBC : Placebo, n = 11; Pasteurized, n =11; Alive, n=8. All tests were performed in two-tailed. *P < 0.05.

Figure 2. Changes in parameters related to lipid metabolism.

(a) Total cholesterol, (b) LDL-cholesterol and (c) Triglycerides. Differential values (MD and MD from placebo) are expressed as mean ± SEM, either as raw data or as percentages. Bars represent mean change from baseline value per group, with their standard errors. Mann-Whitney tests or unpaired T-tests were performed to compare differential values of both treated groups versus the Placebo group (inter-group changes), according to the distribution. The respective P value are indicated in the table and when the test is significative, bars are marked by * symbol. Lines represent raw values before and 3 months after receiving treatment. Distribution of values within each group for each timing is illustrated by a box and whisker. In the box plots, the line in the middle of the box is plotted at the median, the inferior and superior limit of the box correspond to the 25th and the 75th percentiles respectively. The whiskers correspond to the minimum and maximum value. Wilcoxon matched-pairs signed ranks tests or Paired T-tests were performed to verify changes from baseline (intra-group changes), according to the distribution,when drawn, the capped line above the concerned group shows the corresponding P-value. When the difference is significative, a capped line is marked above the concerned group. Changes between 0 and 3 months across the 3 groups were analyzed with Kruskal-Wallis or One-way ANOVA according to the distribution and group-wise comparisons were performed using Bonferroni and Tuckey’s adjustment for multiple testing, respectively. When the difference is significative, a line is marked above the concerned groups with the corresponding P value. MD, Mean Difference. Placebo, n = 11; Pasteurized, n =12; Alive, n=9 for all parameters. All tests were performed in two-tailed. *P < 0.05.

Figure 3. Changes in hepatic and general enzymes.

(a) Alanine aminotransferase activity (ALT), (b) Aspartate aminotransferase activity (AST), (c) ɣ-Glutamyltransferase (GGT), (d) Lipopolysaccharides (LPS), (e) Lactate Dehydrogenase (LDH) and (f) Creatine Kinase (CK). Differential values (MD and MD from placebo) are expressed as mean ± SEM, either as raw data or as percentages. Bars represent mean change from baseline value per group, with their standard errors. Mann-Whitney tests were performed to compare differential values of both treated groups versus the Placebo group (inter-group changes), according to the distribution. The respective P value are indicated in the table and when the test is significative, bars are marked by * symbol. Lines represent raw values before and after 3 months of supplementation. Distribution of values within each group for each timing is illustrated by a box and whisker. In the box plots, the line in the middle of the box is plotted at the median, the inferior and superior limit of the box correspond to the 25th and the 75th percentiles respectively. The whiskers correspond to the minimum and maximum value. Wilcoxon matched-pairs signed ranks tests were performed to verify changes from baseline (intra-group changes), according to the distribution. When the difference is significative, a capped line is marked above the concerned group with the corresponding P value. Kruskal-Wallis analysis were used to compare changes between 0 and 3 months across the 3 groups according to the distribution. All group-wise comparisons were performed using Bonferroni’s adjustment for multiple testing. When the difference is significative, a line is marked above the concerned groups with the corresponding P value. MD, Mean Difference. Placebo, n = 11; Pasteurized, n =12; Alive, n=9 for all parameters except for Creatine Kinase : Placebo, n = 11; Pasteurized, n =11; Alive, n=8. All tests were performed in two-tailed. *P < 0.05.

Figure 4. Changes in anthropometric parameters.

(a) Body weight, (b) Fat mass, (c) Hip circumference and (d) Waist circumference. Differential values (MD and MD from placebo) are expressed as mean ± SEM, either as raw data or as percentages. Bars represent mean change from baseline value per group, with their standard errors. Mann-Whitney tests were performed to compare differential values of both treated groups versus the Placebo group (inter-group changes), according to the distribution. The respective P value are indicated in the table. Lines represent raw values before and after 3 months of supplementation. Distribution of values within each group for each timing is illustrated by a box and whisker. In the box plots, the line in the middle of the box is plotted at the median, the inferior and superior limit of the box correspond to the 25th and the 75th percentiles respectively. The whiskers correspond to the minimum and maximum value. Wilcoxon matched-pairs signed ranks tests were performed to verify changes from baseline (intra-group changes), according to the distribution and, when drawn, the capped line above the concerned group shows the corresponding P-value. Kruskal-Wallis analysis was used to compare changes between 0 and 3 months across the 3 groups according to the distribution. All group-wise comparisons were performed using Bonferroni’s adjustment for multiple testing. MD, Mean Difference. Placebo, n = 11; Pasteurized, n =12; Alive, n=9 for all parameters except for Hip and Waist: Placebo, n = 10; Pasteurized, n =12; Alive, n=9. All tests were performed in two-tailed. MD, Mean Difference. All tests were performed in two-tailed.