Metformin Modulates Ferroptosis-Related and Antioxidant Gene Expression in Brown Adipose Tissue

Metformin is a widely prescribed antidiabetic agent with pleiotropic effects extending beyond glycemic control, including anti-inflammatory and antioxidant actions. However, its impact on cytokine signaling and ferroptosis-related pathways in brown adipose tissue (BAT) remains poorly characterized. Here, we examined the transcriptional and metabolic responses to metformin in brown adipocytes and in a diet-induced obesity model. Differentiated brown adipocytes were treated with metformin and analyzed by RNA sequencing and RT-qPCR. C57BL/6 male mice fed a high-fat diet (HFD) were administered metformin to assess systemic metabolic parameters and BAT-specific responses, with validation by histology, RT-qPCR, and Western blot. Transcriptomic profiling identified differentially expressed genes enriched in cytokine-cytokine receptor interaction and ferroptosis-related KEGG pathways. Metformin modulated the expression of multiple inflammatory cytokine genes and upregulated antioxidant and ferroptosis defense-related genes, including the glutathione biosynthesis genes Gclc and Gclm, together with Hmox1, Gpx4, Nfe2l2 (Nrf2), and Slc7a11. Among these, Gpx4 showed the most consistent upregulation across mRNA and protein levels in BAT. In HFD-fed mice, metformin improved glucose tolerance and elevated the expression of antioxidant and ferroptosis-related genes in BAT, consistent with the in vitro findings. Together, these results indicate that metformin coordinately modulates antioxidant and ferroptosis defense-related gene expression in BAT, suggesting a tissue-protective transcriptional program under metabolic stress. Our findings identify candidate immunometabolic and ferroptosis-related targets of metformin in BAT and provide a basis for further mechanistic investigation of its tissue-specific actions beyond glycemic control.