2 Type 2 Diabetes,Metformin,and Gut Microbiota Diabetes Car (11-13).a mucin-degrading bacteria nt.and individuals diae their associated genes(microbiome) with Alzheimer disease,Parkinson dis ute the T our env In numans,parti r any other ne rative d ved to deterministic role in had similar abundance of Subdoliaran and gastrointestinal diseases (crohn dis lum and Akkerm ansi ease.ulcerative colitis short bo wel syn of type .or diah globe,has been linked in nonhuman (1) may help ameliorate a type 2 diabetes dance with the principles of the Dec ation of Helsink the nost rerent hu takir elative abundance of Adlercreutzia(17). and 008430 of 1993).All of the partici don was bn a poten metage ants were th nt (6) and pron e assured of anonymity and conf Metformi 1.1 imethylbigua dia ontrast tiality nfor wa th ith with had Th (7).and findings from recent studi dance of fubacte nmittee of Sede de cio t ma o pre 53 17 min has nleiot ic effe vet the Subdoli anulum and a and an ved the r s de echa nist cluster of butyrate- oducing Clostr 588 ings pro alters hepatic glucose production via to the antidiabe effects of metformin metric,Clinical,and Dietary Evalu through 03 that mucin we BM (kg)/heigh esi the gut (11 n this lean (18.5 BMI ke/m 509% eralizability of ght B/m ng t inte inal an -30 21 ation of metformin is 300 times that of of HDI IDI VIDI total choles dysbiosis in a colombian adult popula iglycerides,apolipoprotein B asting the plasma (16),mal on.Given the conside ariation in etes and that the obtained (collection and m administration of metformin lombians is differen to tha of othe nt explained in Supplemen y Data a14 518 hyp n the 24-h 。e throug ns a Data) al me ormin a rogates in s ferent to those obs and 2 ction and enc effect of metfo nin enrichment of mucin-degrading and scFA Each participant collected their own fe- ela producing micro ealed,ste the RESEARCH DESIGN AND METHODS receptacle ffect than imm r ext Study Design erated in h sehold freezers and brough e July and N ember 2 ty in ea xposure than the other metformin for ld with BMI =18 5 kg/m2 living in sively in the morning (6 AM-12 PM) mulations(15 the Colom of M n,bt ted the anima 11 2,1 16171 nlled in the dav ay that metformin may partially restore sured by the health insu rance prov ide samples were stored on dry ice and ith type next-da formin treatment increased the relativ individual who consu anti evaluated by trained lab abundance of Akkermansia muciniphilo biotics or antiparasitics <3 months prior oratory technicians. Microbial communities (microbiota) and their associated genes (microbiome) constitute the interface of our environs and our cells, and their composition is believed to play a deterministic role in human health and disease. In particular, the development of type 2 diabetes, a disease rising in prevalence around the globe, has been linked in nonhuman (1) and human (2–5) studies to imbalances in microbiota of the intestinal tract (gut). However, the most recent human study on this topic found that the asso￾ciation was modulated in a potent￾ially beneficial manner by metformin treatment (6). Metformin (1,1-dimethylbiguanide hydrochloride) is the most frequent medication used to treat type 2 diabetes (7), and findings from recent studies suggest it may also prevent cancer (7) and cardiovascular events (8). Metfor￾min has pleiotropic effects, yet the majority of mechanistic studies have focused on changes in liver function (7,9,10). Although metformin certainly alters hepatic glucose production via effects on AMPK, there is growing evi￾dence that the genesis of its action is in the gut (11–15). Metformin is ;50% bioavailable, al￾lowing for near-equal intestinal and plasma exposure, but intestinal accumu￾lation of metformin is 300 times that of the plasma (16), making the gut the pri￾mary reservoir for metformin in humans. Unlike oral administration, intravenous administration of metformin in humans does not improve glycemia (14). More￾over, in mice, oral administration of a broad-spectrum antibiotic cocktail with oral metformin abrogates metformin’s glucose-lowering effect (12). Providing yet further evidence that the glucose￾lowering effect of metformin may originate in the lower bowel, a delayed-release oral metformin, which targets the ileum, had a similar or greater glucose-lowering effect than immediate-release or extended￾release metformin, despite the delayed￾release metformin having lower systemic exposure than the other metformin for￾mulations (15). Recent studies in animals (11,12,13) and humans (6,17) provide evidence that metformin may partially restore gut dysbiosis associated with type 2 di￾abetes. In mice fed a high-fat diet, met￾formin treatment increased the relative abundance of Akkermansia muciniphila (11–13), a mucin-degrading bacteria that has been shown to reverse meta￾bolic disorders (1,12). In humans, partic￾ipants with diabetes taking metformin had similar abundance of Subdoligranu￾lum and, to some extent, Akkermansia compared with control subjects without diabetes, suggesting that metformin may help ameliorate a type 2 diabetes– associated gut microbiome (6). It has also been shown that people with diabetes taking metformin had a higher relative abundance of Adlercreutzia (17), and metagenomic functional analyses dem￾onstrated significantly enhanced butyrate and propionate production in people with diabetes using metformin (6). In contrast, people with diabetes who were not treat￾ed with metformin had a higher abun￾dance of Eubacterium and Clostridiaceae SMB53 (17) and lower levels of short￾chain fatty-acid (SCFA)–producers, such as Roseburia, Subdoligranulum, and a cluster of butyrate-producing Clostri￾diales (6). These findings provide evi￾dence that gut microbes may contribute to the antidiabetes effects of metformin through pathways that include mucin degradation and SCFA production. In this study we aimed to test the gen￾eralizability of previous observations con￾cerning the influence of metformin on the association of type 2 diabetes and gut dysbiosis in a Colombian adult popula￾tion. Given the considerable variation in the microbiota associated with type 2 di￾abetes and that the gut microbiota of Co￾lombians is different to that of other populations (18), we hypothesized that the microbial taxa involved in the type 2 diabetes dysbiosis of Colombians are dif￾ferent to those observed in Chinese and European populations (2,3) but that the effect of metformin is similar, i.e., through enrichment of mucin-degrading and SCFA￾producing microbiota. RESEARCH DESIGN AND METHODS Study Design Between July and November 2014, we en￾rolled 459 men and women 18–62 years old, with BMI $18.5 kg/m2 , living in the Colombian cities of Medellin, Bogota, Barranquilla, Bucaramanga, and Cali. All participants enrolled in the study were in￾sured by the health insurance provider EPS y Medicina Prepagada Suramericana S.A. (EPS SURA). We excluded pregnant women, individuals who consumed anti￾biotics or antiparasitics ,3 months prior to enrollment, and individuals diagnosed with Alzheimer disease, Parkinson dis￾ease, or any other neurodegenerative dis￾eases; current or recent cancer (,1 year); and gastrointestinal diseases (Crohn dis￾ease, ulcerative colitis, short bowel syn￾drome, diverticulosis, or celiac disease). This study was conducted in accor￾dance with the principles of the Decla￾ration of Helsinki, as revised in 2008, and had minimal risk according to the Colombian Ministry of Health (Resolution 008430 of 1993). All of the partici￾pants were thoroughly informed about the study and procedures. Participants were assured of anonymity and confi- dentiality. Written informed consent was obtained from all the participants before beginning the study. The Bioethics Committee of Sede de Investigacion´ UniversitariadUniversity of Antioquia re￾viewed the protocol and the consent forms and approved the procedures de￾scribed here (approbation act 14–24–588 dated 28 May 2014). Anthropometric, Clinical, and Dietary Evaluations We calculated BMI as weight (kg)/height squared (m2 ) to classify participants as lean (18.5 # BMI , 25.0 kg/m2 ), over￾weight (25.0 # BMI , 30.0 kg/m2 ), or obese (BMI $30 kg/m2 ). In addition, val￾ues of HDL, LDL, VLDL, total cholesterol, triglycerides, apolipoprotein B, fasting glucose, glycated hemoglobin (HbA1c), fasting insulin, adiponectin, and hs-CRP were obtained (collection and measure￾ment explained in Supplementary Data). Dietary intake was evaluated through 24-h dietary recalls (see Supplementary Data). DNA Extraction and Sequence Analysis Each participant collected their own fe￾cal sample in a hermetically sealed, ster￾ile receptacle provided by the research team. Samples were immediately refrig￾erated in household freezers and brought to an EPS SURA facility in each city within 12 h; receipt of samples occurred exclu￾sively in the morning (6 A.M.–12 P.M.). As such, stools were collected between the evening of the day before and the morn￾ing of the day of sample receipt. Fecal samples were stored on dry ice and sent to a central laboratory via next-day delivery. Before DNA extraction, stool consistency was evaluated by trained lab￾oratory technicians. 2 Type 2 Diabetes, Metformin, and Gut Microbiota Diabetes Care