Metformin preconditioning protects Daphnia pulex from lethal hypoxic insult involving AMPK, HIF and mTOR signaling

Hypoxic tolerance is subjected to modification during environmental stress. Understanding the mechanisms underlying the plasticity of hypoxic tolerance will be helpful for clinical applications such as stroke prevention. In a freshwater invertebrate, Daphnia pulex, we found that preconditioning with the antidiabetic drug metformin protects the animals from hypoxic insult. Metformin is known to activate the cellular energy sensor, AMP-activated protein kinase (AMPK). Application of the AMPK antagonist, compound C, effectively abolished the protective action by metformin. Meanwhile, the AMPK agonist AICAR failed to mimic the protective effect of metformin. At transcript level, metformin treatment increased the expression of hypoxia-inducible factor (HIF) α and β genes, as well as the HIF target genes in an AMPK-dependent manner, while AICAR treatment alone failed to increase the expression of HIF genes. Metformin treatment also increased the expression of AMPK α and γ genes, and this effect was blocked by compound C. These observations suggest that HIF activation and HIF target gene expression are possibly involved in metformin-mediated protection, while AMPK activation is necessary, but not sufficient for metformin-induced protection. Since increased hypoxic tolerance involves regenerative responses and thus protein synthesis, we measured the gene expression of the components of mTOR signaling pathway. Metformin increased the gene expression of raptor, a component of mTORC1 and known to control protein synthesis, and such increase was also eliminated by compound C. Taken together, metformin preconditioning activates multiple signaling pathways involved in gene expression and protein synthesis.