ORIGINAL ARTICLE

An increase in the Akkermansia spp. populationinduced by metformin treatment improves glucosehomeostasis in diet-induced obese miceNa-Ri Shin,1 June-Chul Lee,2 Hae-Youn Lee,2 Min-Soo Kim,1 Tae Woong Whon,1

Myung-Shik Lee,2 Jin-Woo Bae1

▸ Additional material ispublished online only. To viewplease visit the journal online(http://dx.doi.org/10.1136/gutjnl-2012-303839).1Department of Life andNanopharmaceutical Sciencesand Department of Biology,Kyung Hee University, Seoul,Korea2Department of Medicine andDepartment of Biotechnology& Bioengineering, SamsungMedical Center, SungkyunkwanUniversity School of Medicine,Seoul, Korea

Correspondence toDr Jin-Woo Bae,Department of Life andNanopharmaceutical Sciencesand Department of Biology,Kyung Hee University, Seoul130-701, Korea; baejw@khu.ac.kror Dr Myung-Shik Lee,Department of Medicine,Samsung Medical Center,Sungkyunkwan UniversitySchool of Medicine, Seoul 135-710, Korea; mslee0923@skku.edu

N-RS, J-CL and H-YLcontributed equally to thiswork.

Received 28 September 2012Revised 15 May 2013Accepted 30 May 2013Published Online First26 June 2013

▸ http://dx.doi.org/10.1136/gutjnl-2013-305370

To cite: Shin N-R, Lee J-C,Lee H-Y, et al. Gut2014;63:727–735.

ABSTRACTBackground Recent evidence indicates that thecomposition of the gut microbiota contributes to thedevelopment of metabolic disorders by affecting thephysiology and metabolism of the host. Metformin isone of the most widely prescribed type 2 diabetes (T2D)therapeutic agents.Objective To determine whether the antidiabetic effectof metformin is related to alterations of intestinalmicrobial composition.Design C57BL/6 mice, fed either a normal-chow diet or ahigh-fat diet (HFD), were treated with metformin for6 weeks. The effect of metformin on the composition ofthe gut microbiota was assessed by analysing 16S rRNAgene sequences with 454 pyrosequencing. Adipose tissueinflammation was examined by flow cytometric analysis ofthe immune cells present in visceral adipose tissue (VAT).Results Metformin treatment significantly improved theglycaemic profile of HFD-fed mice. HFD-fed mice treatedwith metformin showed a higher abundance of the mucin-degrading bacterium Akkermansia than HFD-fed controlmice. In addition, the number of mucin-producing gobletcells was significantly increased by metformin treatment(p<0.0001). Oral administration of Akkermansiamuciniphila to HFD-fed mice without metforminsignificantly enhanced glucose tolerance and attenuatedadipose tissue inflammation by inducing Foxp3 regulatoryT cells (Tregs) in the VAT.Conclusions Modulation of the gut microbiota (by anincrease in the Akkermansia spp. population) may contributeto the antidiabetic effects of metformin, thereby providing anew mechanism for the therapeutic effect of metformin inpatients with T2D. This suggests that pharmacologicalmanipulation of the gut microbiota in favour of Akkermansiamay be a potential treatment for T2D.

INTRODUCTIONWorldwide prevalence of obesity has increased overthe past 30–40 years owing to changes in dietary pat-terns and reduced physical activity.1 The conditionhas now become a pandemic.2 3 Although theprimary reason for obesity is an imbalance betweendietary energy intake and expenditure, it is becomingclear that the gut microbiota plays a role in control-ling energy balance and immune homeostasis.4–11

The human gut microbiota comprises 10–100 trillionmicroorganisms and more than 1000 different bac-terial species.12 It also plays important roles in regu-lating the host’s metabolism and extracting energyfrom ingested food.4 8 10 13

Apart from its beneficial functions for the host,gut microbiota can potentially take part in patho-physiological interactions with the host, particularly

Significance of this study

What is already known about this subject?▸ Alterations in the gut microbiota composition

play a role in the development of adiposetissue inflammation and in the pathogenesis ofobesity-induced diabetes.

▸ The presence of the mucin-degradingbacterium, Akkermansia spp., is associatedwith healthy mucosa.

▸ Immune cells resident in the adipose tissue arecritical promoters of obesity and insulinresistance.

What are the new findings?▸ The microbial community in the intestine of

metformin-treated high-fat diet-fed (HFD-Met)mice exhibited a unique microbial consortiumthat was distinct from that in the HFD-fedcontrol mice (HFD-CT) or normal-chow diet(NCD)-fed mice (NCD-CT and NCD-Met).

▸ Marked changes in the abundance of 29genera, particularly mucin-degradingAkkermansia, accounted for the observeddifferences in the gut microbial communitiesfollowing a HFD or metformin treatment.

▸ The number of mucin-producing goblet cellsincreased upon metformin treatment, regardlessof the dietary composition or glucose tolerance.

▸ Akkermansia-administered HFD-fed mice(HFD-Akk) showed improved glucose toleranceand an increase in the number of goblet cellsand adipose tissue-resident CD4 Foxp3regulatory T cells (Tregs).

How might it impact on clinical practice inthe foreseeable future?▸ This study provides circumstantial evidence that

modulation of the gut microbial community,either by metformin treatment or byAkkermansia administration, may result in animproved metabolic profile in patients withtype 2 diabetes.

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in the case of obesity and related metabolic disorders. Recentstudies have shown that changes in the gut microbiota may playa role in the pathogenesis of the obese and diabetic phenotypes.For example, germ-free mice are protected from high-fat diet(HFD)-induced obesity and metabolic dysfunction, includingglucose intolerance.4 6 In addition, colonisation of germ-freeanimals with gut microbiota isolated from conventionally raisedobese donors led to a significant increase in body fat contentand insulin resistance in recipient mice.10 14 Similarly, the trans-fer of gut microbiota from Toll-like receptor 5-deficient mice towild-type germ-free mice also transferred the diabetic pheno-type of the Toll-like receptor 5-deficient donor to the recipi-ent.15 Taken together, these studies suggest that alterations inthe gut microbial community increase the capacity of the host toextract energy from a given diet, thereby triggering the develop-ment of obesity and diabetes. This indicates a link between thegut microbiota and the diabetic phenotype of the host.

Obesity and type 2 diabetes (T2D) are also associated withchronic low-grade tissue inflammation.16 17 Numerous studiessuggest that adipose tissue is not just a fat storage depot; it alsocontrols systemic metabolism by acting as an endocrine organ.18

Moreover, inflammation within adipose tissue disrupts triglycer-ide storage, and the subsequent excess of free fatty acids inducesinsulin resistance.19 At the cellular level, immune cells presentin adipose tissue, such as neutrophils and macrophages, partici-pate in obesity-induced pathology.20 21 Of the different types ofimmune cells that reside in adipose tissue, CD4 Foxp3 regula-tory T cells (Tregs) control local inflammation by counteractingthe infiltration of M1 macrophages and CD8 T cells, therebypreventing adipose tissue inflammation.22 The protective roleplayed by these Tregs has also been demonstrated in severalanimal models of obesity and insulin resistance, such as leptin-deficient mice and HFD-fed mice.23 Both the percentage andnumber of Tregs in the adipose tissue of obesity-induced dia-betic animals are considerably lower than those in lean controls.

Metformin (1,1-dimethylbiguanide hydrochloride) has beenwidely used to treat T2D for the past 50 years.24–26 In T2D,increased hepatic glucose production is an important cause ofhyperglycaemia. Although the precise mechanism underlyingthe glucose-lowering effect of metformin is not fully under-stood, the most commonly accepted mechanism involves thesuppressed transcription of genes involved in gluconeogenesisvia activation of AMP-activated protein kinase (AMPK), anenzyme that senses cellular energy levels and regulates the avail-ability of fuel.27–29 The intestinal microbiota may also controlthe activation status of AMPK. For example, germ-free micethat are protected against diet-induced obesity show increasedlevels of AMPK activity in the liver and in muscle tissue.6

Furthermore, the intestines themselves make an important con-tribution to the glucose-lowering effect of metformin.30–32

Several studies report much higher concentrations of metforminin the intestinal mucosa than in other tissues.31 32 These find-ings raise the possibility that metformin may have both directand indirect effects on the gut microbiota, which may in turncontribute to its antidiabetic effects.

However, no experimental or clinical studies have examinedthe effects of antidiabetic drugs on the gut microbiota, althoughgut microbial dysbiosis has been reported in patients withT2D.33 34 Therefore, questions about whether metformin, as anantidiabetic agent, regulates glucose metabolism by modulatingthe gut microbiota remain unanswered. This study examined thetherapeutic effect of metformin on the progression of the dia-betic phenotype in HFD-induced obese and diabetic mousemodels and studied the possible contribution of the gut

microbiota. The results show that metformin induces a pro-found shift in the composition of the gut microbiota communityand suggest a mechanism by which this drug exerts its thera-peutic effect in patients with diabetes.

MATERIALS AND METHODSAnimalsC57BL/6 mice (4-week-old, n=24; Charles River Laboratories,L’Arbresle, France) were maintained in groups of no more thansix mice per cage with free access to food and water. Mice werefed either a normal-chow diet (NCD) or a HFD (60% fat, 20%protein, 20% carbohydrate (kcal/100 g), #D12492; ResearchDiets, New Brunswick, New Jersey, USA). After 8 weeks, themice were divided into four groups of six and fed the followingdiets: (1) a NCD without metformin treatment (NCD-CT), (2)a NCD with metformin treatment (NCD-Met), (3) a HFDwithout metformin treatment (HFD-CT) or (4) a HFD withmetformin treatment (HFD-Met). The two groups ofmetformin-treated mice (NCD-Met and HFD-Met) received300 mg/kg/day of metformin by oral gavage for a period of6 weeks. Body weight was monitored once a week. Akkermansiamuciniphila MucT (CIP107 961) were cultured anaerobically onbrain–heart infusion (BBL) agar plates containing porcine mucin(Sigma) at 37°C. The bacteria were harvested at the late expo-nential growth phase, suspended in thioglycolate–phosphate-buffered saline (PBS) (4.0×108 cfu) and orally administered toHFD-fed mice (HFD-Akk; n=6).

Biochemical analysesGlucose homeostasisMice were fasted overnight and a glucose tolerance test was per-formed after an intraperitoneal injection of glucose (1 g/kg bodyweight). The homeostasis model assessment of insulin resistance(HOMA-IR) index was calculated as previously described.35

Blood glucose concentrations were measured with anAccu-Check glucometer (Roche) both before (0 min) and after(15, 30, 60, 120 and 180 min) the glucose injections.

HistologyThe distal ileum was removed from all the mice, flushed withPBS, fixed in 4% formaldehyde overnight at room temperatureand paraffin-embedded. The specimens were then cut into 5 μmsections and stained with Alcian blue periodic acid–Schiff (PAS)(the goblet cells were stained blue). The results were expressedas the number of goblet cells per intestinal villus.

Analysis of gut microbiota and quantification ofAkkermansia muciniphilaThe methods used to analyse the gut microbiota and to quantifyA muciniphila are described in the online supplementarymethods (see online supplementary table S1).

Isolation of stromal vascular fraction cells and flowcytometryThe visceral adipose tissue (VAT) was excised and minced intosmall pieces (>2 mm) and incubated for 20 min in a collagenasesolution (1 mg/mL of collagenase type 2 (Worthington) inDulbecco’s modified Eagle’s medium). The digested tissue wascentrifuged at 1000g for 8 min. The pellet containing thestromal vascular fraction (SVF) was resuspended in PBS and fil-tered through a 70 μm mesh filter. The cells were then washedwith PBS, incubated in red blood cell (RBC) lysis buffer (Sigma)for 3 min and resuspended in fluorescence-activated cell sorting(FACS) buffer (2 mM EDTA, 2% fetal bovine serum in PBS).

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The isolated cells were then incubated with labelled monoclonalantibodies (eBioscience and BD Pharmingen) and analysed byflow cytometry using a FACSAria flow cytometer (BectonDickinson). Data were analysed with FlowJo software (Tree Star,Inc). We validated the flow cytometric identification of CD11cF4/80 CD11b (M1) and CD206 F4/80 CD11b (M2) adiposetissue macrophages. The percentage of Tregs was analysed bythree-colour flow cytometry after staining with antibodies toCD3, CD4 and mouse/rat Foxp3 (FJK-16 s), as previouslydescribed.36

Statistical analysisData were expressed as the mean±SEM. Significance of the dif-ferences between the groups of mice was assessed using theStudent t test. For experiments comparing multiple groups, thedifferences were analysed by one-way analysis of variance fol-lowed by Duncan’s post-hoc test. p Values <0.05 were consid-ered significant. The nearest shrunken centroid (NSC) methodwas used to detect the bacterial genera that were specificallyover- or under-represented within each category (diet, treatmentor diet–treatment combinations).37 The amount of shrinkagewas chosen to minimise the overall misclassification error. Theseanalyses allowed the identification of bacterial genera whoserelative abundance was significantly different between the cat-egories.38 The analysis was performed using the PredictiveAnalysis for Microarrays package under R software.

RESULTSMetformin improves glucose homeostasis under HFDconditionsTo assess the effect of metformin on obesity and glucose intoler-ance, we used a glucose tolerance test to examine glucosehomeostasis in metformin-treated and non-treated mice fedeither a NCD or a HFD. As expected, in comparison withNCD-CT mice, HDF-CT mice showed an increase in the areaunder the curve (AUC), an increased HOMA-IR index and anincreased incidence of fasting hyperglycaemia (figure 1), all ofwhich suggest glucose intolerance. Total body weight and fatpad weight also increased in mice fed a HFD (see online supple-mentary figure S1). Compared with HFD-CT mice, HFD-Metmice showed a significant improvement in glucose tolerance,fasting blood glucose levels and the HOMA-IR index (p<0.01)(figure 1A–D), demonstrating that metformin has an anti-hyperglycaemic effect. Metformin treatment did not affectfasting blood glucose levels, glucose tolerance or the HOMA-IRindex in NCD-fed mice. In contrast to its effects on glucosemetabolism, 6 weeks of metformin treatment had no effect onbody weight or fat pad weight in NCD- or HFD-fed mice (seeonline supplementary figure S1), indicating that the improve-ments in glucose tolerance seen in the HFD-Met mice are notrelated to changes in body weight or fat mass.

Effect of metformin on the composition of the gut microbialcommunitySince the composition of the gut microbiota is associated withT2D,33 we next determined the effect of metformin on thecomposition of the gut microbiota using metagenomic analysisbased on 454 pyrosequencing technology. A total of 121 051raw reads were obtained from 20 fecal samples. After perform-ing quality control procedures to remove low-quality sequences,63 132 sequences (with an average length of 370±25 bases)were subjected to further analysis (see online supplementarytable S2).

Classification of the sequences was carried out by non-OTU(operational taxonomic units)-based assessment using theRibosomal Database Project classifier. All the sequences wereassigned to eight bacterial phyla, comprising three phyla thatdominate the distal gut in both mice and humans: Firmicutes,Bacteroidetes and Actinobacteria.8 Although all individuals areborn with a specific gut microbiome, dietary factors have a sig-nificant influence on the development of the gut microbial com-position.8 10 13 We observed that HFD-CT mice had a greaterabundance of Firmicutes (p<0.001) and a lower abundance ofBacteroidetes (p<0.001) than NCD-CT mice (see online supple-mentary figure S2A). In addition, HFD-CT mice showed a sig-nificantly lower abundance of the phyla Verrucomicrobia(p<0.001) and TM7 (p<0.01) than NCD-CT mice.

Thus, we hypothesised that metformin may alter the compos-ition of the gut microbiota in parallel with its antidiabetic effecton host glucose metabolism. Metformin treatment resulted in aprofound shift in the fecal microbial community profiles of thegut microbiota in diabetic mice (see online supplementary figureS2B). The tendency toward phyla-wide changes after metformintreatment was similar in NCD- and HFD-fed mice; however,the changes were more marked in the HFD-fed group. Therewere significant differences in the abundance of Firmicutes andBacteroidetes between HFD-CT mice and HFD-Met mice(p<0.001), but there was no significant difference in the abun-dance of these two phyla between NCD-Met mice andNCD-CT mice. Importantly, the abundance of Verrucomicrobiawas significantly increased in the HFD group after metformintreatment (p<0.001), whereas no significant change was seen inthe NCD group. This suggests that metformin-induced modula-tion of the gut microbiota is diet-dependent.

The overall composition of the bacterial community in thedifferent groups was characterised by quantifying and interpret-ing similarities based on phylogenetic distances using UniFracmetric. The principal coordinates analysis of UniFrac-based pair-wise comparisons of community structures disclosed an unex-pected distribution of the microbial community among the fourgroups of mice. The main finding of the principal coordinatesanalysis was that different diets promote the development of dif-ferent gut microbial communities. Furthermore, HFD-Met miceformed a cluster that was distinct from that of HFD-CT miceand not even intermediate between those of the controlHFD-fed and NCD-fed mice (figure 2). The microbial commu-nities of the NCD-fed groups were, however, closely clustered,even in mice treated with metformin; this indicates that metfor-min has a more marked effect on gut microbial communitycomposition in HFD-fed mice than it does in NCD-fed mice.Taken together, these results indicate that metformin has differ-ent effects on the gut microbial composition of NCD- andHFD-fed mice.

Next, to determine which bacterial genera contributed tothese differences in microbiota composition between the fourgroups, we performed a NSC analysis. The specifically definedcomparisons between community characteristics were tabulatedon a heat map representing the relative abundance of eachgenus. By combining both statistical and NSC analyses, wefound that changes in the abundance of 29 genera, belonging tosix phyla, accounted for the differences in the gut microbialcommunities seen in mice fed different diet or metformin treat-ment (figure 3), which suggests the possibility that the antidia-betic effect of metformin may be mediated by a specific subsetof bacterial taxa. The relative abundance of Anaerotruncus,Lactococcus, Akkermansia, Parabacteroides, Odoribacter,Alistipes, Lawsonia, Blautia and Lactonifactor was altered by a

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HFD; however, metformin rescued these HFD-inducedchanges, restoring the levels to those seen in NCD-fed mice.Despite the low relative abundance (0.3–2.9%) of sequencesassigned to Akkermansia, this genus made a large contributionto the observed differences in the composition of the gut micro-bial communities in HFD-CT and HFD-Met mice. Moreover,Akkermansia was responsible for the greater abundance ofVerrucomicrobia seen in HFD-Met mice compared withHFD-CT mice. Thus, metformin restored the relative abun-dance of specific bacterial genera in HFD-Met mice to that seenin NCD-fed mice, an action that may play a role in its glucose-lowering effects.

The unexpected changes in the composition of the gut micro-biota in HFD-Met mice prompted us to examine whether deplet-ing the microbiota using broad-spectrum antibiotics wouldabrogate the antidiabetic effects of metformin. To examine this,HFD-fed mice were treated with a combination of antibiotics(carbenicillin, metronidazole, neomycin and vancomycin) beforemetformin treatment. It is noteworthy that HFD-fed micetreated with antibiotics, but not treated with metformin

(HFD-Abx), showed a significant improvement in glucose toler-ance in comparison with HFD-CT mice (p<0.05) (see onlinesupplementary figure S3). However, the reduction in the AUCseen for metformin- and antibiotic-treated HFD-fed mice(HFD-Abx+Met) was not significantly different from that seenfor HFD-Abx mice. This suggests that antidiabetic effect of met-formin can be abrogated in the absence of the gut microbiota.

Metformin treatment increases the number of goblet cellsBecause metformin significantly increased the relative abun-dance of Akkermansia in HFD-Met mice and Akkermansia usesmucus as a nutrient source,39 we next examined the ileum inmice from the different treatment groups and counted thenumber of mucin-producing goblet cells. PAS–Alcian blue stain-ing showed that metformin treatment increased the number ofPAS-positive goblet cells (figure 4A–D). To evaluate thesechanges further, we counted the number of PAS-positive gobletcells per villus. An average of 6.6±0.3 goblet cells was seen inHFD-CT mice, which was lower than that in NCD-CT mice(7.8±0.2) (figure 4E). Interestingly, metformin treatment

Figure 2 Cluster analysis of control (CT) or metformin-treated (Met) mice fed a normal-chow diet (NCD) or a high-fat diet (HFD). Bacterialcommunities were clustered using the principal coordinates analysis (PCoA) of unweighted (A) and weighted (B) UniFrac distance matrices. The firsttwo principal coordinates (PC1 and PC2) from the PCoA of unweighted and weighted UniFrac are plotted for each sample. The percentage variationin the plotted principal coordinates is indicated on the axes. Each spot represents one sample and each group of mice is denoted by a differentcolour.

Figure 1 Metformin improves glucose homeostasis in HFD-fed (diabetic) mice. The glucose tolerance test (GTT) (A), the area under the curve(AUC) for the GTT curves (B), the fasting glucose levels (C) and the homeostasis model assessment of insulin resistance (HOMA-IR) index (D) areshown for both control (CT) and metformin-treated (Met) mice fed a normal-chow diet (NCD) or a high-fat diet (HFD). All mice were fastedovernight (16 h) before the GTT. Data are expressed as the mean±SEM (n=6 mice/group). The letters denote the level of statistical significance(p<0.05) using one-way analysis of variance followed by Duncan’s post-hoc test.

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significantly increased the number of goblet cells in both NCD-and HFD-fed mice (NCD-Met, 9.1±0.2; HFD-Met, 9.5±0.5;p<0.001), indicating that metformin alters the goblet cell popu-lation regardless of metabolic profile or dietary composition. Inaddition, Pearson correlation analysis showed that the numberof goblet cells was positively correlated with the abundance ofAkkermansia (figure 4F).

Administration of A muciniphila improves insulin signallingby inducing VAT-resident CD4 Foxp3 TregsSince we observed both a greater abundance of Akkermansia andan increase in the number of goblet cells in HFD-Met mice, wenext investigated whether a selective increase in the Akkermansiapopulation had any effect on the metabolic profile or the numberof goblet cells in HFD-fed mice. Mice were fed a HFD for

Figure 3 Striking differences in theabundance of the different bacterialgenera that comprise the gut microbialcommunities in NCD-fed and HFD-fedmice in CT and Met mice. (A) Nearestshrunken centroids of the 29 generathat account for the differences in thecomposition of the microbialcommunities between the four groupsof mice. For the genera listed in thecentre, those that are over-representedare designated by rightward-extendingbars. The bars denotingunder-represented genera extend tothe left. The length of the bar indicatesthe strength of the effect. (B) Heatmap showing the relative abundanceof each classified genus. Each columnon the heat map represents a sampleof 20 mice (n=5 mice/group) and eachrow represents the genus listed in thecentre. A range of colours, from yellowto black, indicates the prevalence ofeach genus. All the genera listed in thecentre showed significant differences(p<0.05; one-way analysis of variancefollowed by Duncan’s post-hoc test).NCD-CT, normal-chow diet (NCD)-fedcontrol mice; NCD-Met, NCD-fedmetformin-treated mice; HFD-CT,high-fat diet (HFD)-fed control mice;HFD-Met, HFD-fed metformin-treatedmice.

Figure 4 The goblet cell populationis affected by metformin treatment.(A–D) Representative lightphotomicrographs showing ilealsections stained with periodic acid–Schiff (PAS)/Alcian blue. (A) NCD-CTmice. (B) NCD-Met mice. (C) HFD-CTmice. (D) HFD-Met mice. (E) Changesin goblet cells (blue in A–D) areexpressed as means±SEM of thenumber of PAS-positive goblet cells pervillus. The letters above the barsdenote statistically significantdifferences (p<0.05) between themeans (one-way analysis of variancefollowed by Duncan’s post-hoc test).(F) The relationship between therelative abundance of Akkermansiaand the number of goblet cells(Pearson’s correlation test; r=0.4996,p=0.02). NCD-CT, normal-chow diet(NCD)-fed control mice; NCD-Met,NCD-fed metformin-treated mice;HFD-CT, high-fat diet (HFD)-fed controlmice; HFD-Met, HFD-fedmetformin-treated mice.

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4 weeks, after which they were given 4.0×108 cfu of A mucini-phila orally every day for a further 6 weeks. Colonisation ofAkkermansia was confirmed by calculating the relative fractionalabundance of Akkermansia by quantitative PCR. Despite differ-ences in the initial abundance of Akkermansia between HFD-CTand HFD-Akk mice, Akkermansia treatment prevented anydecrease in abundance in HFD-fed mice as they aged (see onlinesupplementary figure S4). Consistent with the positive correl-ation between the number of goblet cells and the abundance ofAkkermansia, supplementation with Akkermansia restored boththe number (per villus) and density (per unit surface area) ofgoblet cells in HFD-fed mice (figure 5A–B and online supple-mentary figure S5).

Furthermore, HFD-Akk mice showed a significant improve-ment in glucose tolerance (p<0.01) (figure 5C), similar to thatseen in HFD-Met mice. This indicates that Akkermansia hasthe potential to ameliorate the glucose tolerance induced by aHFD. However, treatment with a lower dose (4.0×106 cfu) oflive Akkermansia or with the same dose of autoclaved (dead)cells did not ameliorate the impaired glucose tolerance (seeonline supplementary figure S6), suggesting that the bacteriamust be metabolically active and used at a dose > 4.0×107 cfuto exert their beneficial effect. We next studied the effect ofAkkermansia on metabolic hormones. As expected, metforminsignificantly reduced the increases in serum insulin and leptinlevels induced by a HFD (p<0.001 for insulin; p<0.05 forleptin) (see online supplementary figure S7A and B), an effectthat is probably secondary to improvements in glucose homeo-stasis. Similarly, HFD-Akk mice showed reduced serum concen-trations of insulin and leptin compared with HFD-CT mice,although the differences were not statistically significant (seeonline supplementary figure S7A and B). The HFD-inducedincreases in serum lipopolysaccharide (LPS) levels reduced bymetformin or Akkermansia, suggesting that the decreased endo-toxaemia seen in HFD-Akk mice might be related to improvedglucose tolerance. However, the differences were not statistic-ally significant compared with HFD-CT mice (see online sup-plementary figure S7C). Furthermore, neither metformin norAkkermansia affected intestinal permeability in HFD-fed mice(see online supplementary figure S7D).

Because changes in intestinal permeability cannot explain theimproved metabolic profile seen after Akkermansia administra-tion, we next examined the possibility that Akkermansia mightexert an antidiabetic effect by reducing SVF inflammation in theVAT, which has been implicated in the aetiology of insulin resist-ance.21 40 FACS analysis showed a significant increase in theproportion of M1 macrophages and a decrease in the propor-tion of M2 macrophages in the VATof HFD-CT mice comparedwith that in NCD-CT mice. However, M1/M2 macrophagepolarisation in the SVF of HFD-fed mice was not affected byAkkermansia or metformin (see online supplementary figureS8). In addition, we also observed a significant difference in theCD4/CD8 T cell ratio in the SVF of NCD-CT and HFD-CTmice; however, the CD4/CD8 T cell ratio in the SVF ofHFD-fed mice was not affected by Akkermansia or metformin(see online supplementary figure S9).

We next examined the number of Tregs, which are criticalregulators of immune or inflammatory responses in the adiposetissues of obese animals. We observed a significant reduction inthe proportion of Tregs in the SVF from the VAT of HFD-CTmice compared with that in NCD-CT mice (p<0.05) (figure 6Aand C), consistent with previous reports.23 Interestingly, admin-istration of Akkermansia to HFD-fed mice restored the Tregproportion to a level comparable to that in NCD-CT mice(NCD-CT, 49.5±2.3; HFD-CT, 32.5±5.7; HFD-Akk, 52.47±2.176; p<0.01; figure 6A and C). There was also a significantincrease in the overall number of Tregs within the SVF inHFD-Met and HFD-Akk mice compared with that in HFD-CTmice (figure 6B). When we examined the expression of proin-flammatory cytokines that play a direct role in tissue inflamma-tion and insulin resistance, we found that the expression ofinterleukin (IL)-6 and IL-1β mRNA was significantly increasedin HFD-CT mice compared with that in NCD-CT mice;however, after treatment with Akkermansia, the expression ofIL-6 and IL-1β was similar to that in NCD-CT mice (figure 6Dand E). Thus, a single bacterial species, A muciniphila, canattenuate VAT inflammation by inducing the generation of Tregsand suppressing the production of proinflammatory cytokines inthe SVF, thereby leading to increased insulin signalling inHFD-fed mice.

Figure 5 Akkermansia administrationimproves glucose tolerance andmodulates the number of goblet cellsin HFD-fed mice. (A) Changes in gobletcells are expressed as the number ofperiodic acid–Schiff (PAS)-positivegoblet cells per villus. (B)Representative light photomicrographsshowing ileal sections stained withPAS/Alcian blue. (C) Area under thecurve (AUC) obtained after the glucosetolerance test (GTT). The results arerepresentative of three independentexperiments. All data are expressed asthe mean±SEM (n=6 mice/group). pValues were determined using aone-tailed unpaired t test. *p<0.05;**p<0.01; ***p<0.001. NCD-CT,normal-chow diet (NCD)-fed controlmice; HFD-CT, high-fat diet (HFD)-fedcontrol mice; HFD-Met, HFD-fedmetformin-treated mice; HFD-Akk,HFD-fed Akkermansia-administeredmice.

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DISCUSSIONThe gut microbiota is highly associated with obesity and T2D.8–10 33

Metformin is one of the most common drugs used to treat patientswith T2D.25 41 Here, we used a metagenomic approach to demon-strate a role for metformin in modulating the composition of the gutmicrobiota in diet-induced obese and diabetic C57BL/6 mice.Consistent with previous findings,7 8 10 42 a HFD led to dysbiosis,increased fasting glucose levels and glucose intolerance. Metformintreatment significantly improved the hyperglycaemia in HFD-CTmice; however, the gut microbiota of HFD-Met mice showed analtered microbial composition, which was distinct from that inHFD-CTor NCD-CT mice. Based on these results, we suggest thatthe gut microbiota might be a contributing factor to the glucose-lowering effect of metformin.

Depending on the diet fed to the mice, metformin had differ-ent effects on microbial composition. The gut microbial com-munity in HFD-Met mice was distinct from that in NCD-CTand NCD-Met mice (figure 2). Specifically, NSC analysis identi-fied several genera that contributed to the observed differencesin the distribution of bacterial communities between the fourgroups (figure 3). Metformin offset the changes in the propor-tions of several genera that were induced by a HFD. Forexample, the significant reductions in the proportions ofAkkermansia and Alistipes and the increases in the proportionsof Anaerotruncus, Lactococcus, Parabacteroides, Odoribacter,Lawsonia, Blautia and Lactonifactor, seen in HFD-CT mice,were reversed by metformin. Consequently, the proportions ofthese genera were similar in HFD-Met and NCD-CT mice (thenon-diabetic phenotype). Depleting the gut microbiota using acombination of antibiotics before metformin treatment abro-gated the antidiabetic effects of the drug, as indicated by thefinding that HFD-Abx+Met mice did not show any additional

reduction in the AUC over that seen in HFD-Abx mice.However, both HFD-Abx and HFD-Abx+Met mice showed asignificant improvement in glucose tolerance compared withHFD-CT mice. Accordingly, recent studies suggest that antibio-tics improve glucose homeostasis of HFD-fed mice.43 44

Furthermore, it is not possible to test whether the antidiabeticeffects of metformin are abrogated in germ-free animals becauseHFD-induced dysbiosis is likely to be responsible for impairedglucose homeostasis.4 6 Despite this, our study supports thehypothesis that changes in the composition of the gut micro-biota induced by antidiabetic drugs contribute to the improvedglucose homeostasis seen in diabetic mice.

Recently, several studies have highlighted the effects of mucusdegradation on host physiology.45 46 The mucin-degrading bac-terium, A muciniphila, which belongs to the phylumVerrucomicrobia, is more abundant in the mucosa of healthysubjects than in that of diabetic patients or animals.47–50 Also,the colonisation of germ-free mice with A muciniphila led toaltered host mucosal transcriptome profiles, which showedbalanced immune responses indicative of tolerance to com-mensal bacteria.46 We observed that a HFD reduced the abun-dance of Akkermansia, suggesting an unhealthy state in thesemice. The abundance of Akkermansia in HFD-Met mice was,however, significantly higher than that in HFD-CT mice, sug-gesting increased caecal mucin levels.45 In this regard, weobserved a greater number of mucin-producing goblet cells inboth NCD- and HFD-fed mice after metformin treatment. Wealso noted a positive correlation between the abundance ofAkkermansia and the number of PAS-positive goblet cells (figure4). Additionally, the amelioration of glucose intolerance and anincrease in the goblet cell number were seen in HFD-Akk mice(figure 5). Akkermansia is a mucin-degrading bacterium and the

Figure 6 The induction of Tregs anda reduction in the levels ofproinflammatory cytokines attenuatevisceral adipose tissue (VAT)inflammation in Akkermansiatreatment to HFD-fed mice. (A)Percentage of Foxp3 Tregs within theCD3 CD4 T cell population in thestromal vascular fraction (SVF) of theVAT. (B) Absolute number of CD3 CD4Foxp3 Tregs per million cells in the SVFof the VAT. (C) Representativefluorescence activated cell sorting plotsshowing the gated Foxp3 Tregs withinthe total SVF CD4 T cell population inthe VAT of each mouse. (D and E)Relative expression of interleukin (IL)-6and IL-1β mRNA in the VAT. All dataare expressed as the mean±SEM (n=6mice/group). p Values were determinedusing a one-tailed unpaired t test.*p<0.05; **p<0.01; ***p<0.001.NCD-CT, normal-chow diet (NCD)-fedcontrol mice; HFD-CT, high-fat diet(HFD)-fed control mice; HFD-Met,HFD-fed metformin-treated mice;HFD-Akk, HFD-fedAkkermansia-administered mice.

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proportion of Akkermansia correlates with an increase in thenumber of goblet cells; thus, an increase in the number ofgoblet cells may underlie the improved glucose profiles seenafter Akkermansia administration.

In addition to producing components of the mucus layer,goblet cells also produce molecules that are associated withinnate defence mechanisms, such as intestinal trefoil factor andresistin-like molecule β. Because these molecules augment barrierintegrity,51 52 an increase in the number of goblet cells afterAkkermansia administration may attenuate glucose intolerance byreducing LPS translocation across the intestinal barrier. However,serum LPS levels and gut permeability were not significantly dif-ferent in HFD-Akk and HFD-CT mice, suggesting that factorsother than changes in barrier function are responsible for theimproved metabolic profile found in HFD-Akk mice.

Of the various T cell subsets, Tregs play a key role in control-ling the classic adaptive immune responses and also the innateimmune responses associated with obesity-induced chronicinflammation.53 In this context, we observed a significantdecrease in the Treg population in HFD-CT mice compared withNCD-CT mice. However, oral administration of Akkermansia toHFD-fed mice restored both the percentage and absolutenumber of Tregs within the total CD4 T cell population in theVAT, which was similar to the effect of metformin treatment(figure 6A–C). These results provide evidence that a selectiveincrease in the Akkermansia population improves the metabolicprofile of individuals with diet-induced obesity via a mechanismthat is mediated by the anti-inflammatory activity of VAT-residentTregs. In addition, the increases in IL-6 and IL-1β mRNA expres-sion seen in the VATafter a HFD were abolished by Akkermansiatreatment (figure 6D and E). This is consistent with the findingsof previous studies suggesting that Akkermansia has anti-inflammatory activity.48 54 Recent papers report that goblet cellsaffect intestinal immune homeostasis by delivering antigens fromthe lumen to tolerogenic dendritic cells, which then induceTregs.55 Thus, the increase in the number of goblet cells afterAkkermansia treatment might also induce Tregs; however, theincreased goblet cell population does not appear to modulate sys-temic LPS or intestinal permeability. Also, the increased gobletcell population might expedite tolerogenic dendritic cellresponses to Akkermansia under HFD conditions and dendriticcell-augmented Treg activity might suppress inflammatorychanges in adipose tissues, leading to increased glucose tolerance.

In summary, our results show that metformin treatmentinduced an unexpected change in the composition of the gutmicrobiota in mice receiving a HFD, which may constitute a newmechanism for the antidiabetic activity of metformin. Moreover,these findings reinforce the concept that changes in the gutmicrobial community, aimed at increasing the Akkermansia popu-lation, can rescue the glucose intolerance associated with T2D.Although it is still unclear which of the molecular mechanisms orinteractions associated with Akkermansia (and which governglucose homeostasis) are involved in increasing the goblet celland Treg populations, our pharmacological intervention studyprovides circumstantial evidence that modulation of the gutmicrobial community improves the health of patients with T2D.It also supports the suggestion that Akkermansia has a potentialrole as a probiotic with antidiabetic effects. It would be interest-ing to undertake a follow-up study to identify the immunomodu-latory molecule(s) and/or soluble factors produced byAkkermansia and their related interactions.

Contributors N-RS, J-CL and H-YL contributed equally to this work. N-RS, J-CLand H-YL performed the experiments and data analysis. N-RS, J-CL, M-SL and J-WB

planned the experiments. N-RS, J-CL, M-SK, TWW, M-SL and J-WB wrote themanuscript. M-SL and J-WB conceived and supervised the study.

Funding This work was supported by a grant from the Mid-Career ResearcherProgram (2011–0028854 to J-WB) through the National Research Foundation ofKorea (NRF), funded by the Ministry of Education, Science and Technology (MEST)and by the Bio R&D Program (2008–04090 to M-SL). M-SL is the recipient of aglobal research laboratory grant from the National Research Foundation of Korea(K21004000003-12A0500-00310).

Competing interests None.

Provenance and peer review Not commissioned; externally peer reviewed.

REFERENCES1 Kahn SE, Hull RL, Utzschneider KM. Mechanisms linking obesity to insulin

resistance and type 2 diabetes. Nature 2006;444:840–6.2 Rokholm B, Baker JL, Sorensen TI. The levelling off of the obesity epidemic since

the year 1999–a review of evidence and perspectives. Obes Rev 2010;11:835–46.3 Wang Y, Beydoun MA, Liang L, et al. Will all Americans become overweight or

obese? estimating the progression and cost of the US obesity epidemic. Obesity(Silver Spring) 2008;16:2323–30.

4 Bäckhed F, Ding H, Wang T, et al. The gut microbiota as an environmental factorthat regulates fat storage. Proc Natl Acad Sci USA 2004;101:15718–23.

5 Bäckhed F, Ley RE, Sonnenburg JL, et al. Host-bacterial mutualism in the humanintestine. Science 2005;307:1915–20.

6 Bäckhed F, Manchester JK, Semenkovich CF, et al. Mechanisms underlying theresistance to diet-induced obesity in germ-free mice. Proc Natl Acad Sci USA2007;104:979–84.

7 Cani PD, Bibiloni R, Knauf C, et al. Changes in gut microbiota control metabolicendotoxemia-induced inflammation in high-fat diet-induced obesity and diabetes inmice. Diabetes 2008;57:1470–81.

8 Ley RE, Bäckhed F, Turnbaugh P, et al. Obesity alters gut microbial ecology. ProcNatl Acad Sci USA 2005;102:11070–5.

9 Ley RE, Turnbaugh PJ, Klein S, et al. Microbial ecology: human gut microbesassociated with obesity. Nature 2006;444:1022–3.

10 Turnbaugh PJ, Ley RE, Mahowald MA, et al. An obesity-associated gut microbiomewith increased capacity for energy harvest. Nature 2006;444:1027–31.

11 Tilg H. Obesity, metabolic syndrome and microbiota: multiple interactions. J ClinGastroenterol 2010;44(Suppl 1):S16–18.

12 Dethlefsen L, Huse S, Sogin ML, et al. The pervasive effects of an antibiotic on thehuman gut microbiota, as revealed by deep 16S rRNA sequencing. PLoS Biol2008;6:e280.

13 Jumpertz R, Le DS, Turnbaugh PJ, et al. Energy-balance studies reveal associationsbetween gut microbes, caloric load and nutrient absorption in humans. Am J ClinNutr 2011;94:58–65.

14 Turnbaugh PJ, Bäckhed F, Fulton L, et al. Diet-induced obesity is linked to markedbut reversible alterations in the mouse distal gut microbiome. Cell Host Microbe2008;3:213–23.

15 Vijay-Kumar M, Aitken JD, Carvalho FA, et al. Metabolic syndrome and altered gutmicrobiota in mice lacking Toll-like receptor 5. Science 2010;328:228–31.

16 Guilherme A, Virbasius JV, Puri V, et al. Adipocyte dysfunctions linking obesity toinsulin resistance and type 2 diabetes. Nat Rev Mol Cell Biol 2008;9:367–77.

17 Xu H, Barnes GT, Yang Q, et al. Chronic inflammation in fat plays a crucial role inthe development of obesity-related insulin resistance. J Clin Invest2003;112:1821–30.

18 Winer S, Winer DA. The adaptive immune system as a fundamental regulator ofadipose tissue inflammation and insulin resistance. Immunol Cell Biol2012;90:755–62.

19 Furuhashi M, Fucho R, Gorgun CZ, et al. Adipocyte/macrophage fatty acid-bindingproteins contribute to metabolic deterioration through actions in both macrophagesand adipocytes in mice. J Clin Invest 2008;118:2640–50.

20 Elgazar-Carmon V, Rudich A, Hadad N, et al. Neutrophils transiently infiltrateintra-abdominal fat early in the course of high-fat feeding. J Lipid Res2008;49:1894–903.

21 Weisberg SP, McCann D, Desai M, et al. Obesity is associated with macrophageaccumulation in adipose tissue. J Clin Invest 2003;112:1796–808.

22 Liu G, Ma H, Qiu L, et al. Phenotypic and functional switch of macrophagesinduced by regulatory CD4+CD25+ T cells in mice. Immunol Cell Biol2011;89:130–42.

23 Feuerer M, Herrero L, Cipolletta D, et al. Lean, but not obese, fat is enriched for aunique population of regulatory T cells that affect metabolic parameters. Nat Med2009;15:930–9.

24 Cusi K, DeFronzo RA. Metformin: A review of its metabolic effects. DiabetesReviews 1998;6:89–131.

25 Donnelly LA, Doney AS, Hattersley AT, et al. The effect of obesity on glycaemicresponse to metformin or sulphonylureas in Type 2 diabetes. Diabet Med2006;23:128–33.

26 Witters LA. The blooming of the French lilac. J Clin Invest 2001;108:1105–7.

734 Shin N-R, et al. Gut 2014;63:727–735. doi:10.1136/gutjnl-2012-303839

Gut microbiota

group.bmj.com on April 7, 2014 - Published by gut.bmj.comDownloaded from

27 Shaw RJ, Lamia KA, Vasquez D, et al. The kinase LKB1 mediates glucosehomeostasis in liver and therapeutic effects of metformin. Science2005;310:1642–6.

28 He L, Sabet A, Djedjos S, et al. Metformin and insulin suppress hepaticgluconeogenesis through phosphorylation of CREB binding protein. Cell2009;137:635–46.

29 Zhou G, Myers R, Li Y, et al. Role of AMP-activated protein kinase in mechanism ofmetformin action. J Clin Invest 2001;108:1167–74.

30 Bailey CJ, Mynett KJ, Page T. Importance of the intestine as a site ofmetformin-stimulated glucose utilization. Br J Pharmacol 1994;112:671–5.

31 Wilcock C, Bailey CJ. Accumulation of metformin by tissues of the normal anddiabetic mouse. Xenobiotica 1994;24:49–57.

32 Bailey CJ, Wilcock C, Scarpello JHB. Metformin and the intestine. Diabetologia2008;51:1552–3.

33 Larsen N, Vogensen FK, van den Berg FW, et al. Gut microbiota in human adultswith type 2 diabetes differs from non-diabetic adults. PLoS One 2010;5:e9085.

34 Qin J, Li Y, Cai Z, et al. A metagenome-wide association study of gut microbiota intype 2 diabetes. Nature 2012;490:55–60.

35 Cai D, Yuan M, Frantz DF, et al. Local and systemic insulin resistance resulting fromhepatic activation of IKK-beta and NF-kappaB. Nat Med 2005;11:183–90.

36 Kim DH, Lee JC, Kim S, et al. Inhibition of autoimmune diabetes by TLR2 tolerance.J Immunol 2011;187:5211–20.

37 Koren O, Spor A, Felin J, et al. Human oral, gut and plaque microbiota in patientswith atherosclerosis. Proc Natl Acad Sci USA 2011;108(Suppl 1):4592–8.

38 Knights D, Kuczynski J, Koren O, et al. Supervised classification of microbiotamitigates mislabeling errors. ISME J 2011;5:570–3.

39 Derrien M, Vaughan EE, Plugge CM, et al. Akkermansia muciniphila gen. nov., sp.nov., a human intestinal mucin-degrading bacterium. Int J Syst Evol Microbiol2004;54:1469–76.

40 Nishimura S, Manabe I, Nagasaki M, et al. CD8+ effector T cells contribute tomacrophage recruitment and adipose tissue inflammation in obesity. Nat Med2009;15:914–20.

41 Bailey CJ, Turner RC. Metformin. N Engl J Med 1996;334:574–9.42 Everard A, Lazarevic V, Derrien M, et al. Responses of gut microbiota and glucose

and lipid metabolism to prebiotics in genetic obese and diet-induced leptin-resistantmice. Diabetes 2011;60:2775–86.

43 Carvalho BM, Guadagnini D, Tsukumo DM, et al. Modulation of gut microbiota byantibiotics improves insulin signalling in high-fat fed mice. Diabetologia2012;55:2823–34.

44 Membrez M, Blancher F, Jaquet M, et al. Gut microbiota modulation withnorfloxacin and ampicillin enhances glucose tolerance in mice. FASEB J2008;22:2416–26.

45 Van den Abbeele P, Gerard P, Rabot S, et al. Arabinoxylans and inulin differentiallymodulate the mucosal and luminal gut microbiota and mucin-degradation inhumanized rats. Environ Microbiol 2011;13:2667–80.

46 Derrien M, Van Baarlen P, Hooiveld G, et al. Modulation of Mucosal ImmuneResponse, Tolerance and Proliferation in Mice Colonized by the Mucin-DegraderAkkermansia muciniphila. Front Microbiol 2011;2:166.

47 Liou AP, Paziuk M, Luevano JM Jr., et al. Conserved shifts in the gut microbiotadue to gastric bypass reduce host weight and adiposity. Sci Transl Med2013;5:178ra41.

48 Png CW, Linden SK, Gilshenan KS, et al. Mucolytic bacteria with increasedprevalence in IBD mucosa augment in vitro utilization of mucin by other bacteria.Am J Gastroenterol 2010;105:2420–8.

49 Santacruz A, Collado MC, Garcia-Valdes L, et al. Gut microbiota composition isassociated with body weight, weight gain and biochemical parameters in pregnantwomen. Br J Nutr 2010;104:83–92.

50 Swidsinski A, Dorffel Y, Loening-Baucke V, et al. Acute appendicitis is characterisedby local invasion with Fusobacterium nucleatum/necrophorum. Gut 2011;60:34–40.

51 Artis D, Wang ML, Keilbaugh SA, et al. RELMbeta/FIZZ2 is a goblet cell-specificimmune-effector molecule in the gastrointestinal tract. Proc Natl Acad Sci USA2004;101:13596–600.

52 Suemori S, Lynch-Devaney K, Podolsky DK. Identification and characterization of ratintestinal trefoil factor: tissue- and cell-specific member of the trefoil protein family.Proc Natl Acad Sci USA 1991;88:11017–21.

53 Cipolletta D, Kolodin D, Benoist C, et al. Tissular T(regs): a unique population ofadipose-tissue-resident Foxp3+CD4+ T cells that impacts organismal metabolism.Semin Immunol 2011;23:431–7.

54 Hansen CH, Holm TL, Krych L, et al. Gut microbiota regulates NKG2D ligandexpression on intestinal epithelial cells. Eur J Immunol 2013;43:447–57.

55 McDole JR, Wheeler LW, McDonald KG, et al. Goblet cells deliver luminal antigento CD103+ dendritic cells in the small intestine. Nature 2012;483:345–9.

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doi: 10.1136/gutjnl-2012-303839 2014 63: 727-735 originally published online June 26, 2013Gut

 Na-Ri Shin, June-Chul Lee, Hae-Youn Lee, et al. diet-induced obese miceimproves glucose homeostasis inpopulation induced by metformin treatment

spp.AkkermansiaAn increase in the

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