A purified membrane protein from Akkermansia muciniphila or the pasteurized bacterium improves metabolism in obese and diabetic mice

Journal name:
Nature Medicine
Volume:
23,
Pages:
107–113
Year published:
DOI:
doi:10.1038/nm.4236
Received
Accepted
Published online

Obesity and type 2 diabetes are associated with low-grade inflammation and specific changes in gut microbiota composition1, 2, 3, 4, 5, 6, 7. We previously demonstrated that administration of Akkermansia muciniphila to mice prevents the development of obesity and associated complications8. However, the underlying mechanisms of this protective effect remain unclear. Moreover, the sensitivity of A. muciniphila to oxygen and the presence of animal-derived compounds in its growth medium currently limit the development of translational approaches for human medicine9. We have addressed these issues here by showing that A. muciniphila retains its efficacy when grown on a synthetic medium compatible with human administration. Unexpectedly, we discovered that pasteurization of A. muciniphila enhanced its capacity to reduce fat mass development, insulin resistance and dyslipidemia in mice. These improvements were notably associated with a modulation of the host urinary metabolomics profile and intestinal energy absorption. We demonstrated that Amuc_1100, a specific protein isolated from the outer membrane of A. muciniphila, interacts with Toll-like receptor 2, is stable at temperatures used for pasteurization, improves the gut barrier and partly recapitulates the beneficial effects of the bacterium. Finally, we showed that administration of live or pasteurized A. muciniphila grown on the synthetic medium is safe in humans. These findings provide support for the use of different preparations of A. muciniphila as therapeutic options to target human obesity and associated disorders.

At a glance

Figures

  1. Pasteurization enhances A. muciniphila-mediated effects on high-fat diet-induced obesity.
    Figure 1: Pasteurization enhances A. muciniphila–mediated effects on high-fat diet-induced obesity.

    (a,b) Body weight gain (a) and fat mass gain (b) in grams after 4 weeks of treatment. (c) Daily food intake per mouse in grams. (d) Plasma glucose (mg dl−1) profile and (e) the mean area under the curve (AUC) measured during an oral tolerance test (OGTT) (mg dl−1 min−1). (f) Plasma insulin (μg l−1) measured30 min before and 15 min after glucose administration during the OGTT. (g) Insulin resistance index. (h) Ileum goblet cell density. Data are presented as the mean ± s.e.m. Number of mice per group for a,b: ND: 9, HFD: 8, HFD live Akk mucus: 9, HFD live Akk synthetic: 10, HFD pasteurized Akk: 8. For c, 5 measurements were obtained for each group. Number of mice per group for dg: ND: 9, HFD: 8, HFD live Akk mucus: 9, HFD live Akk synthetic: 10, HFD pasteurized Akk: 7. Number of mice per group for h: ND: 7, HFD: 8, HFD live Akk mucus: 8, HFD live Akk synthetic: 8, HFD pasteurized Akk: 7. Data were analyzed using one-way ANOVA followed by Tukey post hoc test for ac,e,g,h, and according to two-way ANOVA followed by Bonferroni post hoc test for d,f. *P < 0.05; **P < 0.01; ***P < 0.001.

  2. Pasteurized A. muciniphila modulates adipose tissue physiology, intestinal energy absorption and urinary metabolome.
    Figure 2: Pasteurized A. muciniphila modulates adipose tissue physiology, intestinal energy absorption and urinary metabolome.

    (a) Representative hematoxylin and eosin (H&E)-stained pictures of subcutaneous adipose tissue (SAT) deposits (n = 5 images per mouse). Scale bars, 100 μm. (b) Mean adipocyte diameter (μm) in the SAT. (c) Plasma leptin (ng ml−1). (d) Fecal energetic content (kcal g feces−1). (e) Orthogonal partial least-squares discriminant analysis (OPLS-DA) predictive score plot for urine metabolic profiles representing predictive component 1 (Tpred1) versus Tpred2. (f) Projection of all treatment groups on the first predictive score of the OPLS-DA model. (g) Empirical assessment of the significance of O-PLS goodness-of-fit parameters. (h,i) Relative abundance of urinary (h) trimethylamine (TMA) and (i) trimethylamine-N-oxide (TMAO). (j) mRNA expression of hepatic flavin-containing monooxygenase 3. (k) Plasma TMA (μM). (l) Plasma TMAO (μM). Data are presented as the mean ± s.e.m. Number of mice per group for ac and jl: ND: 10, HFD: 8, HFD live Akk synthetic: 10, HFD pasteurized Akk: 9. For d, 5 measurements were obtained for each group. Number of mice per group for ei: ND: 5, HFD: 7, HFD live Akk synthetic: 5, HFD pasteurized Akk: 5. Data were analyzed using one-way ANOVA followed by a Tukey post hoc test for bd, and hk. *P < 0.05; ** P < 0.01; ***P < 0.001.

  3. A.muciniphila protein Amuc_1100[ast] recapitulates the effects of the pasteurized bacterium on diet-induced obesity.
    Figure 3: A.muciniphila protein Amuc_1100* recapitulates the effects of the pasteurized bacterium on diet-induced obesity.

    (ad) Stimulation of human HEK-Blue cells expressing (a) human TLR2, (b) TLR5, (c) TLR9 and (d) human NOD2 receptor. (e) Dynamic light scattering analysis of Amuc_1100* folding state according to the temperature. (f,g) Total body weight gain (f) and total fat mass gain (g) in grams after 5 weeks of treatment. (h) Daily food intake per mouse (g). (i) Plasma VLDL, LDL and HDL cholesterol (mg dl−1). (j) Plasma triglycerides (mg dl−1). (k) Plasma glucose (mg dl−1) profile and (l) mean area under the curve (AUC) measured during an oral tolerance test (mg dl−1 min−1). (m) Representative western blot of four total western blots for hepatic p-IRβ and β-actin with or without insulin stimulation. Ratio of the vehicle- and insulin-stimulated p-IRβ on the loading control measured by densitometry. (n) Representative western blot, of four total, for hepatic p-Aktthr308, p-Aktser473 and β-actin with or without insulin stimulation. Ratio of the vehicle- and insulin-stimulated p-Aktthr308 and p-Aktser473 to the loading control measured by densitometry. Full-length blots are shown in Supplementary Figures 6 and 7. Data are presented as the mean ± s.e.m. Data in a,c,d represent three independent experiments, except for low concentrations of Amuc_1100* (0.05 and 0.5 μg/ml), where two independent experiments were performed. Data in b represent two independent experiments. Number of mice per group for f,g,i,k,l: ND: 9, HFD: 8, HFD live Akk synthetic: 8, HFD pasteurized Akk: 10, HFD Amuc_1100*: 10. For h, five measurements were obtained for each group. Number of mice per group for j: ND: 9, HFD: 8, HFD live Akk synthetic: 8, HFD pasteurized Akk: 10, HFD Amuc_1100*: 9. Number of mice per group for m,n: ND: 8, HFD: 9, HFD live Akk synthetic: 9, HFD pasteurized Akk: 9, HFD Amuc_1100*: 9. Data were analyzed using one-way ANOVA followed by Dunnett post hoc test versus DMEM condition for a,c,d, using Kruskal-Wallis followed by Dunn post hoc test versus DMEM condition for b, according to one-way ANOVA followed by Tukey post hoc test for fh,j,l, and according to two-way ANOVA followed by Bonferroni post hoc test for i,k,m,n.*P < 0.05; **P < 0.01; ***P < 0.001.

  4. Effects of A. muciniphila or Amuc_1100[ast] on the intestinal barrier function.
    Figure 4: Effects of A. muciniphila or Amuc_1100* on the intestinal barrier function.

    (a) Portal plasma LPS (EU/ml). (b) Expression of Cnr1, Cldn3 and Ocln in the jejunum. (c) Expression of Cnr1, Cldn3 and Ocln in the ileum. (d) Expression of Lyz1, DefA, Reg3g and Pla2g2 in the jejunum. (e) Expression of Lyz1, DefA, Reg3g and Pla2g2 in the ileum. Data are presented as the mean ± s.e.m. Number of mice per group for a: ND: 8, HFD: 8, HFD live Akk synthetic: 5, HFD pasteurized Akk: 8, HFD Amuc_1100*: 9. Number of mice per group for b and d: ND: 8, HFD: 7, HFD live Akk synthetic: 8, HFD pasteurized Akk: 10, HFD Amuc_1100*: 9. Number of mice per group for c and e: ND: 9, HFD: 7, HFD live Akk synthetic: 8, HFD pasteurized Akk: 9, HFD Amuc_1100*: 9. Data were analyzed using one-way ANOVA followed by Tukey post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001.

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Author information

  1. Present address: Metapopulation Research Centre, University of Helsinki, Helsinki, Finland.

    • Noora Ottman
  2. These authors contributed equally to this work.

    • Amandine Everard,
    • Céline Druart &
    • Clara Depommier

Affiliations

  1. Université catholique de Louvain, Louvain Drug Research Institute, WELBIO (Walloon Excellence in Life sciences and BIOtechnology), Metabolism and Nutrition Research Group, Brussels, Belgium.

    • Hubert Plovier,
    • Amandine Everard,
    • Céline Druart,
    • Clara Depommier,
    • Matthias Van Hul,
    • Lucie Geurts,
    • Nathalie M Delzenne &
    • Patrice D Cani
  2. Division of Computational and Systems Medicine, Department of Surgery and Cancer, Imperial College London, London, UK.

    • Julien Chilloux,
    • Antonis Myridakis &
    • Marc-Emmanuel Dumas
  3. Laboratory of Microbiology, Wageningen University, Wageningen, the Netherlands.

    • Noora Ottman,
    • Kees C H van der Ark,
    • Steven Aalvink,
    • Clara Belzer &
    • Willem M de Vos
  4. Institute of Metabolic and Cardiovascular Diseases, I2MC, Inserm, UMR 1048, Toulouse, France.

    • Thibaut Duparc,
    • Laeticia Lichtenstein &
    • Laurent O Martinez
  5. RPU Immunobiology, Department of Bacteriology & Immunology, University of Helsinki, Helsinki, Finland.

    • Judith Klievink,
    • Arnab Bhattacharjee &
    • Willem M de Vos
  6. Pole of Endocrinology, Diabetes and Nutrition, Institut de Recherche Expérimentale et Clinique IREC, Cliniques Universitaires Saint-Luc, Université catholique de Louvain, Brussels, Belgium.

    • Dominique Maiter,
    • Audrey Loumaye,
    • Michel P Hermans &
    • Jean-Paul Thissen

Contributions

P.D.C. and W.M.d.V. conceived the project. P.D.C. supervised the preclinical and clinical aspects, and W.M.d.V. the microbial culturing and expression. P.D.C. and H.P. designed the mouse experiments, performed experiments and interpreted all the results, generated figures and tables and wrote the manuscript; A.E., C. Druart, M.V.H., L.G. and C. Depommier performed experiments. J.C., A.M. and M.-E.D. performed 1H-NMR and UPLC-MS metabolomic analyses. N.M.D. provided reagents and analytic tools. T.D., L.L. and L.O.M. analyzed plasma lipoprotein profiles. C.B., K.C.H.v.d.A., H.P., C. Druart and S.A. performed the culturing and pasteurization of A. muciniphila. J.K. produced and purified Amuc_1100*, which was structurally analyzed by A.B. In vitro analysis of A. muciniphila and Amuc_1100* signaling was carried out by N.O. and C.B. J.-P.T., M.P.H., A.L., D.M., A.E., C. Druart, C. Depommier, W.M.d.V. and P.D.C. designed the clinical study. D.M., A.L., M.P.H. and J.-P.T., screened the subjects and contributed to follow-up. A.E., C. Druart, C. Depommier and P.D.C. followed subjects during the study. All authors discussed results and approved the manuscript.

Competing financial interests

A.E., C. Druart, P.D.C., C.B. and W.M.d.V. are inventors on patent applications dealing with the use of A. muciniphila and its components in the treatment of obesity and related disorders.

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