Abstract
Effects of metformin, the first-line drug for type 2 diabetes therapy, on gut microbiome composition in type 2 diabetes have been described in various studies both in human subjects and animals. However, the details of the molecular mechanisms of metformin action have not been fully understood. Moreover, there is a significant lack of information on how metformin affects gut microbiome composition in female mouse models, depending on sex and metabolic status in well controlled experimental setting. Our study aimed to examine metformin-induced alterations in gut microbiome diversity, composition, and functional implications of high-fat diet-induced type 2 diabetes mouse model, using, for the first time in mice study, the shotgun metagenomic sequencing that allows estimation of microorganisms at species level. We also employed a randomized block, factorial study design, and including 24 experimental units allocated to 8 treatment groups to systematically evaluate the effect of sex and metabolic status on metformin interaction with microbiome. We used DNA obtained from fecal samples representing gut microbiome before and after ten weeks-long metformin treatment. We identified 100 metformin-related differentially abundant species in high-fat diet-fed mice before and after the treatment, with most of the species relative abundances increased. In contrast, no significant changes were observed in control diet-fed mice. Functional analysis targeted to carbohydrate, lipid, and amino acid metabolism pathways revealed 14 significantly altered hierarchies. We also observed sex-specific differences in response to metformin treatment. Males experienced more pronounced changes in metabolic markers, while in females the extent of changes in gut microbiome representatives was more marked, indicated by 53 differentially abundant species with more remarkable Log fold changes compared to the combined-sex analysis. The same pattern manifested regarding the functional analysis, where we discovered 5 significantly affected hierarchies in female groups but not in males. Our results suggest that both sexes of animals should be included in future studies focusing on metformin effects on the gut microbiome.
Highlights
Metformin is the first-line therapy for the treatment of type 2 diabetes (T2D)
We designed this study as a randomized block experiment comprised of three blocks with a three-way factorial treatment arrangement where factors of interest are T2D status induced by high-fat diet (HFD) or control diet (CD) feeding, sex, and metformin therapy status, forming eight different treatment groups – HFD_M_Met, HFD_F_Met, HFD_M_Met+, HFD_F_Met+, CD_M_Met, CD_F_Met, CD_M_Met+, and CD_F_Met+ (Supplementary Figure 1)
All experimental units of the same block were randomly assigned to HFD- or CD-fed groups so that each of the treatment groups would consist of animals with similar body weight, and experimental units of both sexes would be represented in the same number in both types of treatment groups
Summary
Metformin is the first-line therapy for the treatment of type 2 diabetes (T2D). According to the American Diabetes Association and European Association for the study of Diabetes guidelines, it is the preferred option for initiating glucose-lowering due to its efficacy, safety, tolerability, and low cost [1]. The molecular mechanism of action of metformin, remains unclear. There is an increasing evidence that metformin’s action mechanism is associated with physiological processes in the gastrointestinal tract. A more pronounced effect of metformin can be observed when the drug is administered orally than intravenously at an equivalent dose [3]. It has been estimated that 20-30% of people receiving metformin therapy develop gastrointestinal side effects, with approximately 5% being unable to tolerate metformin at all [4]. Metformin accumulates in gastrointestinal tissues [5]; for example, it is 30-300 times more concentrated in the small intestine than in plasma, and 30-50% of the drug reaches the colon and is eliminated with feces [6]
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