care diabetesio als.org de la Cuesta-Zuluaga and Associates 7 0.04 A G H f0.30 0.2 0.1 0.0 -Re of th ng diff ts (logLDA >3).Data p nted n±sE.Ope with ND.D 20- 2-met and NE Prevotello has been associated with A.muciniphila alone may explain the derive from the strengthening of the in carbohydrate degradation beneficial effects of m rmin. estinal mucosal barrie terit of the mucin laver.ther gen that may cause se tions in diabetes demonstrated that stopping ing translocation of proinfammatory 527 ormin treat da ipopolys formin use and eut microbiota com glucag on-like peptide 1(17).A three gucose homeostasis (112).Likewise clmtoCioieiectoalimctagenoic r to T2D s p with SCEA t this bad may bacteria(6).Metformin has also been potentially contributing nce al health in the same way T2D-metparticipants in our study mic effect of metfo min stem from it increases SCFA production through also had higher relative abundance o sition on of t e gut microb ing bac na,but not impacts the metformin alters glucose metabolism metabolism f the microbl ota hosted by through effec on bile aci ( studies have shown that met aforeme uman (11-13)and ment in mice on high-fat diet shifts the human (6,17)studies,metformin use in ticipants),Megasphoera (a bac com rd that o b dy was n g ater re nd Butyri LEfSe biom overy,we also 50n5. mi robiota ave beer fat diets ed with aith rate A.muciniphila or metformin had simila (26,33-35).SCFAs may be beneficial for mprovements in mu dance of the mucin-eading bacteri In-pre and Butyrate,in particu one and glucose tolerance,suggesting that that metformin's health henefits mav lonic epithelium (35).Recent studies inPrevotella has been associated with carbohydrate-based diets and degradation of complex polysaccharides (26), whereas E. casseliflavus is an opportunistic patho￾gen that may cause serious infections in immunosuppressed individuals (27). We found associations between met￾formin use and gut microbiota composi￾tion that were largely consistent whether we compared T2D-met+ participants to ND control subjects or to T2D-met2 par￾ticipants, suggesting that metformin may have direct microbial effects. Our findings are congruent with multiple lines of evi￾dence indicating the gut-mediated glyce￾mic effect of metformin stem from alterations in the gut microbiota compo￾sition. Cabreiro et al. (28) first demon￾strated that metformin impacts the metabolism of the microbiota hosted by Caenorhabditis elegans. Three mouse studies have shown that metformin treat￾ment in mice on high-fat diet shifts the microbiota composition toward that of mice fed normal chow by increasing abundance of Akkermansia spp. (11–13). Moreover, a follow-up experiment by Shin et al. (12) found that mice fed high￾fat diets treated with either cultured A. muciniphila or metformin had similar improvements in mucin-producing gob￾let cells, proinflammatory interleukin-6, and glucose tolerance, suggesting that A. muciniphila alone may explain the beneficial effects of metformin. In human studies, a small nonrandom￾ized clinical trial of 12 patients with type 2 diabetes demonstrated that stopping metformin treatment for 7 days led to alterations in the gut microbiota and glucagon-like peptide 1 (17). A three￾country cross-sectional metagenomic study found that metformin use was pos￾itively associated with SCFA-producing bacteria (6). Metformin has also been shown to enhance active and total glucagon-like peptide 1 (17,29,30), which is consistent with the hypothesis that it increases SCFA production through modification of the gut microbiota com￾position. There is also the possibility that metformin alters glucose metabolism through effects on bile acid secretion (31). As we hypothesized, on the basis of the aforementioned nonhuman (11–13) and human (6,17) studies, metformin use in our study was associated with greater rel￾ative abundance of the mucin-degrading A. muciniphila. Through LEfSe biomarker discovery, we also found metformin was positively associated with the mucolytic bacterium B. bifidum. The higher abun￾dance of the mucin-degrading bacteria A. muciniphila and B. bifidum in the gut microbiota of metformin users suggests that metformin’s health benefits may derive from the strengthening of the in￾testinal mucosal barrier. A. muciniphila plays a crucial role in maintaining the in￾tegrity of the mucin layer, thereby reduc￾ing translocation of proinflammatory lipopolysaccharides and controlling fat storage, adipose tissue metabolism, and glucose homeostasis (1,12). Likewise, B. bifidum can grow on gastric mucin as a sole carbon source, and genome anal￾ysis revealed that this bacterium can use host mucins (32), potentially contributing to gastrointestinal health in the same way as A. muciniphila. T2D-met+ participants in our study also had higher relative abundance of some SCFA-producing bacteria, but not others (e.g., Roseburia, Subdoligranulum, Faecalibacterium). Those positively as￾sociated with metformin use included B. bifidum, Prevotella (an OTU distinct from the OTU enriched in T2D-met2 par￾ticipants), Megasphaera (a bacterium re￾lated to Megamonas), and Butyrivibrio (although this taxa was no longer signifi- cant after adjusting for multiple compar￾isons). These microbiota have been associated with production of SCFAs, in￾cluding butyrate, propionate, and acetate (26,33–35). SCFAs may be beneficial for health. Butyrate, in particular, is one of the preferred energy sources of the co￾lonic epithelium (36). Recent studies in 0 0.01 0.02 0.03 0.04 Relave abundance 0 0.10 0.20 0.30 AB C D E F GH *** *** ** * ** ** ** ** ** * ** ** ** ** + *** * * * * * Figure 3—Relative abundance of the groups of bacteria displaying differences among participants (logLDA .3). Data presented as mean 6 SE. Open bars = T2D-met+ ; gray bars = T2D-met2; black bars = ND. A: OTUs enriched in T2D-met2 compared with ND. B: OTUs enriched in ND compared with T2D-met2. C: OTUs enriched in T2D-met+ compared with ND. D: OTUs enriched in ND compared with T2D-met+ . E: OTUs enriched in T2D-met+ compared with T2D-met2. F: OTUs enriched in T2D-met2 compared with T2D-met+ . G: Eleven pooled OTUs classified as Butyrivibrio enriched in T2D￾met+ compared with T2D-met2 and ND. H: Four pooled OTUs classified as A. muciniphila enriched in T2D-met+ compared with T2D-met2 and ND (note the change in scale). +P , 0.1; *P , 0.05; **P , 0.05 and q value ,0.1; ***P , 0.05 and q value ,0.05. care.diabetesjournals.org de la Cuesta-Zuluaga and Associates 7