Table_2_Metformin Strongly Affects Gut Microbiome Composition in High-Fat Diet-Induced Type 2 Diabetes Mouse Model of Both Sexes.xls

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.

二甲双胍（metformin）作为2型糖尿病治疗的一线用药，其对2型糖尿病患者肠道菌群组成的影响已在多项针对人类受试者与动物的研究中得到阐释。然而，二甲双胍作用的分子机制细节尚未完全阐明。此外，在控制良好的实验环境下，关于二甲双胍如何依据性别与代谢状态影响雌性小鼠模型的肠道菌群组成，相关信息存在显著缺失。本研究旨在探究二甲双胍对高脂饮食诱导的2型糖尿病小鼠模型肠道菌群多样性、组成的改变及其功能意义，本研究首次在小鼠实验中采用鸟枪宏基因组测序（shotgun metagenomic sequencing）技术，该技术可实现微生物的物种水平定量分析。本研究同时采用随机区组析因实验设计，将24个实验单元分配至8个处理组，以系统评估性别与代谢状态对二甲双胍与菌群互作的影响。我们提取了为期10周二甲双胍治疗前后的粪便样本DNA，以此表征治疗前后的肠道菌群组成。我们在高脂饮食喂养的小鼠中，共鉴定出100种与二甲双胍相关的差异丰度物种（differentially abundant species），其中多数物种的相对丰度显著升高。与之相反，正常饮食喂养的小鼠未观察到显著的菌群变化。针对碳水化合物、脂质与氨基酸代谢通路的功能分析显示，共有14个层级的代谢通路发生显著改变。我们还观察到小鼠在二甲双胍治疗响应中存在性别特异性差异：雄性个体的代谢标志物变化更为显著，而雌性个体的肠道菌群代表物种的变化幅度更为突出——相较于混合性别分析，雌性组中有53种差异丰度物种的对数倍变化（Log fold change）更为显著。功能分析也呈现出相同模式：我们在雌性组中发现了5个显著改变的代谢通路层级，而雄性组未出现此类变化。本研究结果提示，未来针对二甲双胍对肠道菌群影响的相关研究，应同时纳入雌雄两种性别的实验动物。