Urolithin A improves insulin resistance by targeting TNF-α and regulating the intestinal microbiota

Urolithin A (UA), an intestinal metabolite of ellagic acid, exhibits significant anti-inflammatory activity. Concurrently, Tumor necrosis factor α (TNF-α) serves as a pivotal inflammatory factor, and induces insulin resistance. This study aims to investigate the ameliorative effect of UA on TNF-α-induced insulin resistance and elucidate its underlying molecular mechanism. Methods and results: Through Western Blot (WB), glucose uptake, and immunocytochemistry (ICC) experiments, it was observed that UA significantly alleviated TNF-α-induced insulin resistance in C2C12 cells. Surface plasmon resonance (SPR) and molecular docking experiments validated the binding of UA to TNF-α with an affinity of 9.626×10^-5M, t which demonstrated UA could directly bind to TNF-α and inhibiting its downstream signaling. These results suggested a potential monoclonal antibody-like mechanism of UA to inhibit TNF-α-induced insulin resistance. Furthermore, we also found the  the substantial inhibitory effect of UA on LPS-induced pro-inflammatory polarization in RAW264.7 cells using WB and ICC experiments. In type 2 diabetes mellitus (T2DM) mouse models, OGTT, ITT, Elisa, WB, and RT-PCR experiments revealed that UA could effectively reduce blood glucose levels and serum concentrations of TNF-α and insulin while enhancing insulin signaling pathway transmission in muscle tissues and upregulating the expression of relevant genes for glycogen synthesis. Additionally, analysis of the intestinal microbiota showed that UA intervention markedly reshaped the composition and structure of the microbial community, reduced the elevated Firmicutes/Bacteroidetes ratio and increased the relative abundance of beneficial genera such as Akkermansia, Dubosiella, and Kurthia, as well as the key species Akkermansia muciniphila, thereby ameliorating HFD/STZ-induced dysbiosis and promoting the functional homeostasis of the intestinal ecosystem in T2DM. Conclusion: UA could directly bind to TNF-α and inhibit its downstream signaling, suggesting a potential monoclonal antibody-like mechanism. UA also exerted inhibitory effects on TNF-α-induced insulin resistance by specifically binding to TNF-α in C2C12 myotubes. We also found that UA could significantly improve insulin resistance, enhanced insulin signaling pathway transmission in muscle tissues, and improved intestinal microbiota dysbiosis.