Metformin-induced changes of the gut microbiota in healthy young men: results of a non-blinded, one-armed intervention study

Abstract

Aims/hypothesis: Individuals with type 2 diabetes have an altered bacterial composition of their gut microbiota compared with non-diabetic individuals. However, these alterations may be confounded by medication, notably the blood-glucose-lowering biguanide, metformin. We undertook a clinical trial in healthy and previously drug-free men with the primary aim of investigating metformin-induced compositional changes in the non-diabetic state. A secondary aim was to examine whether the pre-treatment gut microbiota was related to gastrointestinal adverse effects during metformin treatment.

Methods: Twenty-seven healthy young Danish men were included in an 18-week one-armed crossover trial consisting of a pre-intervention period, an intervention period and a post-intervention period, each period lasting 6 weeks. Inclusion criteria were men of age 18-35 years, BMI between 18.5 kg/m² and 27.5 kg/m², HbA_1c < 39 mmol/mol (5.7%) and plasma creatinine within the normal range. No prescribed medication, including antibiotics, for 2 months prior to recruitment were allowed and no previous gastrointestinal surgery, discounting appendectomy or chronic illness requiring medical treatment. During the intervention the participants were given metformin up to 1 g twice daily. Participants were examined five times in the fasting state with blood sampling and recording of gastrointestinal symptoms. Examinations took place at Frederiksberg Hospital, Denmark before and after the pre-intervention period, halfway through and immediately after the end of intervention and after the wash-out period. Faecal samples were collected at nine evenly distributed time points, and bacterial DNA was extracted and subjected to 16S rRNA gene amplicon sequencing in order to evaluate gut microbiota composition. Subjective gastrointestinal symptoms were reported at each visit.

Results: Data from participants who completed visit 1 (n=23) are included in analyses. For the primary outcome the relative abundance of 11 bacterial genera significantly changed during the intervention but returned to baseline levels after treatment cessation. In line with previous reports, we observed a reduced abundance of Intestinibacter spp. and Clostridium spp., as well as an increased abundance of Escherichia/Shigella spp. and Bilophila wadsworthia. The relative abundance at baseline of 12 bacterial genera predicted self-reported gastrointestinal adverse effects.

Conclusions/interpretation: Intake of metformin changes the gut microbiota composition in normoglycaemic young men. The microbiota changes induced by metformin extend and validate previous reports in individuals with type 2 diabetes. Secondary analyses suggest that pre-treatment gut microbiota composition may be a determinant for development of gastrointestinal adverse effects following metformin intake. These results require further investigation and replication in larger prospective studies.

Trial registration: Clinicaltrialsregister.eu 2015-000199-86 and ClinicalTrials.gov NCT02546050 FUNDING: This project was funded by Danish Diabetes Association and The Novo Nordisk Foundation.

Keywords: Drug therapy; Gut microbiota; Intervention; Metformin; Microbiome; Microbiota; Type 2 diabetes.

Fig. 1

Study design and flow chart. (a) One-armed crossover design with five visits and a total of nine faecal samplings (F1–F9). Metformin intervention was initiated after visit 2, with a gradual increase in dose over 3 weeks, from 500 mg to 2000 mg, to minimise adverse effects. Blood samples were drawn at all five visits. Self-reported gastrointestinal symptoms were evaluated at all visits using a VAS. Anthropometrics measurements were taken every 6 weeks and plasma metformin was measured twice during the intervention period to assess compliance. (b) Flowchart of study. Twenty-nine men underwent screening. Two participants were ineligible for inclusion. Twenty-seven were included in the trial. Three participants dropped out during the run-in period: two dropped out immediately after the screening visit and another dropped out immediately after the first visit, for undisclosed reasons. One participant dropped out during the intervention due to severe gastrointestinal discomfort. One participant dropped out during the post-intervention follow-up, for undisclosed reasons. Twenty-three completed the intervention period, 22 participants completed the follow-up and 25 were included in the analyses. Two participants reduced metformin intake because of side effects. GI, gastrointestinal; p-metformin, plasma metformin

Fig. 2

Metformin-responsive bacterial genera exhibiting a change in relative abundance during the metformin intervention. Boxes represent interquartile range (IQR), with the inner horizontal line representing the median. Whiskers represent values within 1.5 × IQR of the first and third quartiles. Circles represent individual samples with lines connecting samples from the same individual. The purple band represents the pre-intervention mean and 95% confidence limits averaged across the three pre-intervention time points. Diamonds and connecting lines represent mean values, with yellow and green diamonds, respectively, representing nominal (p < 0.05) and false discovery rate adjusted (q < 0.05) significant differences from the averaged pre-intervention mean. The relative abundance at each time point during the intervention was compared with the averaged pre-intervention mean by linear mixed model regression ANOVA. Only genera with a significant change at least at one time point following correction for false discovery rate are presented

Fig. 3

Bacterial genera discriminant for development of gastrointestinal side effects. (a) Participants were divided into two groups based on change in overall self-reported gastrointestinal side effects measured on a VAS from baseline to visit 3 (3 weeks into the metformin intervention). Boxes represent interquartile range (IQR), with the inner horizontal line representing the median, whiskers representing values within 1.5 × IQR of the first and third quartiles and circles representing individual samples. (b) Importance of bacterial genera identified by Boruta feature selection as being discriminant at baseline for development of gastrointestinal discomfort during metformin intervention. Genera are ordered by mean decrease in accuracy from an RF model based on baseline abundances of the 12 discriminant genera fitted using bootstrap resampling. (c) ROC curve representing the ability of the RF model to discriminate between participants who develop gastrointestinal discomfort and those who do not. The shaded area represents the 95% CI; AUC = 0.9