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Oral administration of Akkermansia muciniphila elevates
systemic antiaging and anticancer metabolites
Claudia Grajeda-Iglesias, Sylvère Durand, Romain Daillère, Kristina
Iribarren, Fabien Lemaitre, Lisa Derosa, Fanny Aprahamian, Noélie Bossut,
Nitharsshini Nirmalathasan, Frank Madeo, et al.
To cite this version:
Claudia Grajeda-Iglesias, Sylvère Durand, Romain Daillère, Kristina Iribarren, Fabien Lemaitre, et al..
Oral administration of Akkermansia muciniphila elevates systemic antiaging and anticancer metabo-
lites. Aging, Impact Journals, 2021, 13 (5), pp.6375-6405. �10.18632/aging.202739�. �hal-03195909�
www.aging-us.com 6375 AGING
www.aging-us.com AGING 2021, Vol. 13, No. 5
Research Paper
Oral administration of Akkermansia muciniphila elevates systemic
antiaging and anticancer metabolites
Claudia Grajeda-Iglesias
1,2,3,*
, Sylvère Durand
1,2,3,*
, Romain Daillère
4
, Kristina Iribarren
1,8,9,10
,
Fabien Lemaitre
1,8,9,10
, Lisa Derosa
1,8,9,10
, Fanny Aprahamian
1,2,3
, Noélie Bossut
1,2,3
, Nitharsshini
Nirmalathasan
1,2,3
, Frank Madeo
5,6,7
, Laurence Zitvogel
1,8,9,10
, Guido Kroemer
1,2,3,11,12,13
1
Gustave Roussy Comprehensive Cancer Institute, Villejuif, France
2
Centre de Recherche des Cordeliers, Equipe labellisée Par la Ligue Contre le Cancer, Université de Paris, Sorbonne
Université, Inserm U1138, Institut Universitaire de France, Paris, France
3
Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Center, Université Paris Saclay, Villejuif, France
4
EverImmune, Villejuif, France
5
Institute of Molecular Biosciences, NAWI Graz, University of Graz, Graz, Austria
6
BioTechMed-Graz, Graz, Austria
7
Field of Excellence BioHealth, University of Graz, Graz, Austria
8
Inserm U1015, Villejuif, France
9
Center of Clinical Investigations in Biotherapies of Cancer (CICBT) 1428, Villejuif, France
10
Faculty of Medicine, Université Paris Saclay, Le Kremlin-Bicêtre, France
11
Pôle De Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France
12
Suzhou Institute for Systems Medicine, Chinese Academy of Medical Sciences, Suzhou, China
13
Karolinska Institute, Department of Women’s and Children’s Health, Karolinska University Hospital, Stockholm,
Sweden
*Equal contribution
Correspondence to: Guido Kroemer; email: kroemer@orange.fr
Keywords: Akkermansia muciniphila, microbiota, metabolomics, polyamines, fecal microbial transplantation
Received: January 21, 2021 Accepted: February 19, 2021 Published: March 2, 2021
Copyright: © 2021 Grajeda-Iglesias et al. This is an open access article distributed under the terms of the Creative Commons
Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
ABSTRACT
The presence of Akkermansia muciniphila (Akk) in the human gut is associated with good health, leanness and
fitness. Mouse experimentation has demonstrated positive effects for Akk, which counteracts aging, mediates
antiobesity and antidiabetic effects, dampens inflammation and improves anticancer immunosurveillance.
Clinical trials have confirmed antidiabetic effects for Akk. Here, we investigated the time-dependent effects of
oral administration of Akk (which was live or pasteurized) and other bacteria to mice on the metabolome of the
ileum, colon, liver and blood plasma. Metabolomics was performed by a combination of chromatographic and
mass spectrometric methods, yielding a total of 1.637.227 measurements. Akk had major effects on
metabolism, causing an increase in spermidine and other polyamines in the gut and in the liver. Pasteurized
Akk (Akk-past) was more efficient than live Akk in elevating the intestinal concentrations of polyamines, short-
chain fatty acids, 2-hydroxybutyrate, as well multiple bile acids, which also increased in the circulation. All
these metabolites have previously been associated with human health, providing a biochemical basis for the
beneficial effects of Akk.
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INTRODUCTION
The intestinal microbiota plays a primordial role in
human physiology and pathology [1, 2]. Indeed, the
human body must be conceived as a meta-organism
composed by human cells as well as an overwhelming
majority of microbes in the form of phages, bacteria,
archaea and eukaryotes that colonize all exterior and
interior body surfaces, in particular the gastrointestinal
tract. Thus, the organisms composing the gut flora
outnumber human cells by a factor of 10 as far as the
number of cells is concerned, and by a factor of 100 if
the number of genes encoded by the host and is
inhabitants is calculated [3].
The transition of health to disease is often accompanied
by alterations in the composition of the intestinal
microbiota that shifts from a normal state (eubiosis) to a
pathological state (dysbiosis). Within a complex
ecosystem, such shifts cannot be explained in terms of
simple linear cause-effect relationships. Rather, it
appears that many components of the system, both in
the host and in the gut flora, are simultaneously
impacted, causing alterations in gut permeability as well
as a series of disease-associated features in the host
(with inter alia an increase in systemic inflammation,
metabolic syndrome, reduced immune responses, and a
decrease in organismal fitness) and in the microbiota
(with a loss of overall diversity, a disproportionate
expansion of pathogenic species and a depletion of
health-associated taxa) [4–7].
Notwithstanding these complexities, the transfer of the
intestinal flora by fecal microbial transplantation (FMT)
from humans to mice has established the causal
involvement of intestinal dysbiosis in some diseases.
For example, FMT from obese persons into mice favors
excessive weight gain and diabetes in the latter [8, 9].
Similarly, the transfer of feces from cancer patients that
fail to respond to immunotherapy with immune
checkpoint inhibitors into mice transmits subsequent
immunotherapeutic failure to the rodents [10, 11]. Thus,
anticancer immunocompetence can be transferred across
species barriers from one host to another by FMT.
The aforementioned discoveries have placed the
intestinal microbiota in the limelight of scientific
research, spurring attempts to identify individual
bacterial species or consortia of several microbes that
have a positive impact on health. One prominent
bacterium that has wide pro-health effects is
Akkermansia muciniphila (Akk). Akk is
epidemiologically associated with the consumption of
health-related food items, leanness, exercise, fitness and
healthy aging [7, 10, 12–16]. Its transfer into short-lived
mouse strains extends longevity, supporting that Akk
has an antiaging effect [17]. Preclinical experimentation
supporting its antiobesity and antidiabetic effects
associated with a modulation of the urinary metabolome
[18] has been validated by a successful clinical trial
[19]. In mice, Akk can increase the systemic
concentration of anti-inflammatory factors such as α-
tocopherol and β-sitosterol [20], and stimulates
anticancer immune responses in the context of
immunotherapy targeting the PD-1/PD-L1 interaction
[10]. Mechanistically, it is a matter of debate whether
Akk has to be alive to achieve these effects or whether
it can be pasteurized [19]. A heat-resistant protein
produced by Akk has been shown to mediate antiobesity
and antidiabetic effects through the activation of Toll-
like receptors 2 and 4 [18]. However, the detailed
metabolic effects of Akk have not been studied in detail.
Intrigued by these observations, we decided to
investigate the impact of Akk on metabolism in an
unbiased fashion, by means of mass spectrometric
metabolomics. For this, we transferred different
bacteria, as well as human feces alone or together with
Akk into mice and performed metabolomics analyses of
the ileal and colic content as well as the liver and the
plasma. Here, we report the metabolic effects of live
(Akk) and pasteurized Akk (Akk-past) on these
compartments.
RESULTS
Experimental design
In this study, mice were subjected to a defined sequence
of interventions involving sham gavage with phosphate
buffered saline (PBS), oral administration of broad-
spectrum antibiotics (ATB, a combination of ampicillin,
streptomycin and colistin), FMT from cancer patients
and gavage of a series of distinct bacterial species.
These species were selected because they improve the
anticancer effects of cycloheximide-based chemo-
therapy, as true for Burkholderia cepacia (Brc) and
Enterococcus hirae (Hir) [21, 22]. Bacteroides fragilis
(Frg) was chosen because it improves chemotherapy
with oxaliplatin and immunotherapy with CTLA-4
blockade [23, 24]. In addition, we included
Burkholderia sp. (Bur) and Catenibacterium
mitsuokella (Mit) as controls. These species were
administered after ATB conditioning without prior
FMT, allowing for the spontaneous recovery of the gut
microflora. In contrast, since Akk (a human gastro-
intestinal mucin-loving bacterium) requires the presence
of other bacteria to achieve efficient colonization [25],
Akk was gavaged to mice that had previously received a
FMT from a cancer patient that had not responded to
immunotherapy and whose stools had been screened for
absence of Akk. In a first experiment, only live Akk
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was given to mice, comparing its effects to that of other
bacteria or FMT alone over time on days 3, 7 and 14
after discontinuation of ATB (Figure 1A). In a second
experiment (Figure 1B), live Akk was compared to
Akk-past, based on the observation that pasteurization
actually does not destroy the antiobesity and
antidiabetic effects of Akk [18, 19]. This experiment
was designed to characterize long-term effects of Akk
versus Akk-past with one single time point (day 27).
Both experiments, which involved a minimum of 6
mice per group and time point (total 245 mice) were
followed by the recovery of ileal and colonic content as
well as that of liver and plasma. These samples were
then subjected to metabolite extraction [26], optional
chemical derivatization, different types of (gas or
liquid) chromatography and mass-spectrometric
identification of metabolites, either in a targeted mode
(in which each metabolite is identified based on its
chromatographic characteristics coupled to its exact
mass) or in an untargeted mode (in which each
metabolite is identified as a discernible peak with a
defined mass). After analysis of the samples on three
different mass spectrometers, the data were subjected to
R-based informatics treatments to combine results
obtained by different methods into single files (one for
targeted and one for untargeted results for each sample)
and bioinformatics analysis (Figure 1C). In total,
1.637.227 mass spectrometric measurements were
performed in this study.
Figure 1. Schematic view of the experimental design. (A) Several commensals in mono-associated mice previously decontaminated by
broad spectrum antibiotics (ATB) were administered by oral gavage. Akkermansia muciniphila (Akk) was administered by oral gavage to mice
previously decontaminated by broad spectrum ATB and transplanted with human fecal material (FMT). Continuous ATB was administrated in
the drinking water of the animals. (B) Mice previously decontaminated by broad spectrum ATB and FMT-treated, received Akk or the
pasteurized form of Akk (Akk-past) by oral gavage (versus continuous FMT or ATB). PBS was administered as control (in both, (A, B) designs).
(C) General methodology for metabolomics. The impact of the different treatments on the local (ileal, colon) and distal (liver, plasma)
metabolome was evaluated using gas and liquid chromatography coupled to mass spectrometry allowing to detect a broad range of targeted
and untargeted metabolites. Metabolite extraction was performed on the different organs collected from mice at the experiment day
marked with a red arrow in (A, B). Extracts were processed and analyzed by liquid- and gas-chromatography coupled to mass spectrometry.
Data was merged and analyzed using the GRmeta package in R or a built-in software, Compound Discoverer, for untargeted metabolomics.
ATB, antibiotics; PBS, phosphate buffer saline; Brc, Burkholderia cepacia; Hir, Enterococcus hirae; Frg, Bacteroides fragilis; Bur, Burkholderia
sp.; Mit, Catenibacterium mitsuokella; FMT, fecal microbiota transplant; Akk, Akkermansia muciniphila; Akk-past, pasteurized Akkermansia
muciniphila.
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A gradient of microbial effects from the intestine to
the circulation
In the first experiment (Figure 1A), continuous antibiotic
(ATB) treatment caused a massive depletion of ileal
metabolites (as indicated by the green color in the
heatmap) that was manifest on days 3, 7 and 14 post-
ATB (Figure 2 and Supplementary Figure 1 and
Supplementary Table 1), supporting the importance of
the ileal microbiota for the breakdown of nutrients into
small molecules. Thus, more than half of the metabolites
in the small intestine were significantly reduced in their
abundance after ATB treatment. This strong ATB effect
was also observed for the colic content, in which many
metabolites were reduced while others including several
monosaccharides and amino acids were increased in
their abundance (as indicated by the red color in the
heatmap, Figure 2 and Supplementary Figure 2 and
Supplementary Table 2). Gavage with individual
bacteria (Brc, Bur, Frg, Hir, Mit) or FMT, alone or with
Akk upon ATB discontinuation gradually corrected the
ATB-induced depletion of ileal metabolites, as well as
the shifts in colonic metabolism, over time, thus
allowing the ileal and colic metabolomes to recover to a
state that resembles the basal state (represented by the
PBS control). Very similar tendencies were observed for
the hepatic and plasma metabolomes, though with the
important difference that the concentration of most
metabolites remained close-to-unaltered and only a
minority decreased (and rarely increased) upon ATB
treatment (Supplementary Figure 3). Again, the ATB-
induced changes in the hepatic and plasma metabolomes
were reset to close-to-normal levels by reconstitution of
the intestinal microbiota (Figure 2 and Supplementary
Figures 4, 5 and Supplementary Tables 3, 4).
In conclusion, the composition of the hepatic and
circulating metabolomes is less affected by ATB-
mediated sterilization of the gut than the ileal and colic
metabolome, confirming that the internal milieu is
protected against external perturbations. The effects of
individual bacteria on this system appear discrete.
Akkermansia muciniphila (Akk)-specific effects on
metabolism
In the next step, we compared the effects of Akk plus
FMT with those of FMT alone on the ileal, colic
hepatic and circulating metabolomes, at 3, 7 and 14
days after discontinuation of the initial ATB treatment.
In this comparison (Akk+FMT versus FMT) rather
discrete effects were observed by targeted
metabolomic analysis (Figure 3 and Supplementary
Figures 6–9). Volcano plots revealed a few consistent
early (3 days) changes in the ileal and colic
metabolomes with an Akk-induced increase in
N1,N12-diacetylspermine (in both ileum and colon) as
well as an increase in colic short-chain fatty
Figure 2. Targeted metabolomics analysis was performed on the extracts from ileum, colon, liver, and plasma samples from
mice receiving oral gavages with several commensals or PBS (control), or continuous ATB, at days 3, 7 and 14 after the first
oral gavage. Changes in metabolites relative abundance are illustrated. Ileum and colon showed the strongest treatment-dependent
metabolites variations. Hierarchical clustering (euclidean distance, ward linkage method) of the metabolite abundance is shown. Note that
Supplementary Figures 1, 2, 3 and 5 provide the names of each of the metabolites, for each of the different matrices. The purpose of this
figure is to allow for a direct comparison of the amplitude of the metabolic effects of manipulation of the microbiota. ATB, antibiotics; PBS,
phosphate buffer saline; FMT, fecal microbiota transplant; FC, fold change.
