DataSheet1_Potential benefits of metformin and pioglitazone combination therapy via gut microbiota and metabolites in high-fat diet-fed mice.docx

Metformin and pioglitazone monotherapy have been proven to alter gut microbiota in diabetes and obesity. The present study aimed to investigated whether the combined administration of pioglitazone and metformin achieved superior protective effects on high-fat diet (HFD)-fed obese mice and elucidated its molecular mechanism via the gut microbiota and its metabolites. C57BL/6 males were randomly divided into five groups: the control group, fed a normal control diet; the HFD group, fed an HFD; the metformin monotherapy group, fed an HFD and treated with metformin; the pioglitazone monotherapy group, fed an HFD and treated with pioglitazone; and the combination therapy group, fed an HFD and treated with metformin and pioglitazone combination therapy. The cecal contents were collected for 16S rDNA amplicon sequencing and untargeted metabolomics analysis. The results showed that the combination therapy of metformin and pioglitazone significantly improved insulin sensitivity and glucolipid metabolism in HFD-fed mice. Combination therapy markedly altered gut microbiota by increasing beneficial bacteria, such as Bifidobacterium, Christensenellaceae_R-7_group, Faecalibacterium and Roseburia, and decreasing harmful bacteria, such as Oscillibacter and Eubacterium_xylanophilum_group. Fecal metabolites were significantly changed in the combination therapy group, including a reduction in amino acid metabolism and augmentation of lipid metabolism, such as citrulline, sarcosine, D-glutamine, lipoxin A4, prostaglandin E2, stearidonic acid and lucidenic acid A. These results revealed that combined metformin and pioglitazone therapy had synergistic effects or at least have an additive effect on modifying gut microbiota and metabolites, closely associated with improved glucolipid metabolic parameters in HFD-fed mice, which provides novel evidence and promising targets for metformin and pioglitazone combination therapy in type 2 diabetes.

二甲双胍与吡格列酮的单药治疗已被证实能够改变糖尿病和肥胖症患者的肠道菌群。本研究旨在探究吡格列酮与二甲双胍联合给药是否在喂以高脂饮食（HFD）的肥胖小鼠中实现更优的保护效果，并通过肠道菌群及其代谢产物阐明其分子机制。将C57BL/6雄性小鼠随机分为五组：对照组，喂以正常对照饮食；高脂饮食组，喂以高脂饮食；二甲双胍单药治疗组，喂以高脂饮食并接受二甲双胍治疗；吡格列酮单药治疗组，喂以高脂饮食并接受吡格列酮治疗；联合治疗组，喂以高脂饮食并接受二甲双胍与吡格列酮联合治疗方案。收集盲肠内容物进行16S rDNA扩增子测序和非靶向代谢组学分析。结果显示，二甲双胍与吡格列酮的联合治疗显著改善了喂以高脂饮食的小鼠的胰岛素敏感性和葡萄糖脂质代谢。联合治疗通过增加有益细菌，如双歧杆菌、Christensenellaceae_R-7_group、Faecalibacterium和Roseburia，以及减少有害细菌，如Oscillibacter和Eubacterium_xylanophilum_group，显著改变了肠道菌群。联合治疗组粪便代谢物发生显著变化，包括氨基酸代谢的减少和脂质代谢的增强，如精氨酸、丝氨酸、D-谷氨酰胺、脂氧素A4、前列腺素E2、硬脂酸和木犀草酸A。这些结果揭示了二甲双胍与吡格列酮联合治疗在调节肠道菌群和代谢产物方面具有协同作用或至少具有相加效应，这与改善喂以高脂饮食的小鼠的葡萄糖脂质代谢参数密切相关，为二甲双胍与吡格列酮联合治疗2型糖尿病提供了新的证据和有前景的治疗靶点。