Metformin at environmental relevant concentration induced glucose metabolism disorder in frog: Integrated microstructural, physiological, transcriptomic, and metabolomic insight.

Metformin, a commonly prescribed antidiabetic drug, is increasingly detected in aquatic environments due to its high water solubility and resistance to degradation in conventional wastewater treatment systems. While the ecotoxicological risks of metformin at environmentally relevant concentrations to wild organisms remain unclear. In this study, adult male frog Pelophylax nigromaculatus were exposed to metformin at 0, 10, and 100 µg/L for 21 days to assess hepatotoxicity and metabolic disruption. Histological and biochemical assessments revealed liver injuries, including reduced hepatosomatic index, elevated transaminase levels, and oxidative damage. Integrated transcriptomic and metabolomic analyses identified alterations in glycolysis, gluconeogenesis, pyruvate metabolism, and oxidative phosphorylation. Mechanistically, metformin enhanced glycolysis by upregulating hexokinase, phosphofructokinase, and pyruvate kinase, while suppressing gluconeogenesis via reduced phosphoenolpyruvate carboxykinase and glucose-6-phosphatase expression. These molecular changes correlated with decreased hepatic glycogen and blood glucose, but increased lactate and lactate dehydrogenase activity. Collectively, our results show that environmental concentrations of metformin disrupt amphibian glucose homeostasis by shifting hepatic energy metabolism toward glycolysis and away from gluconeogenesis. This study provides novel mechanistic insight into sub-lethal pharmaceutical risks and underscores the urgency of ecological risk assessments for metformin in freshwater systems.