ETV5 knockdown increases free fatty acid-mediated H2O2 production in pancreatic beta cells but does not affect nitric oxide production and AMPK activation

Introduction The transcription factor ETV5 regulates several obesity- and diabetes-related genes and mediates acinar tissue protection during acute pancreatitis. We previously showed that ETV5-knockdown in beta cells increases lipotoxic stress by palmitate (PA) in association with elevated levels of reactive oxygen species (ROS), but without affecting inflammatory NF-kB activation or ER stress. Here, we investigated the potential role of nitric oxide (NO) and H2O2 for the phenotype, and tested whether ETV5 is involved in the activation of the ROS sensor AMPK, which is a major regulator of antioxidant enzymes.

Methods INS-1E beta cells with doxycycline (Dox)-inducible lentiviral ETV5 shRNA knockdown were exposed for 24 h to 0.4 mM PA and 80 µM H2O2 (LD50 concentrations), and the NO donor DETA NONOate. We assessed viability, NO and ROS by assays. Inducible NO synthase (iNOS) expression was measured by qPCR and AMPK phosphorylation by western blot.

Results ETV5 knockdown significantly increased NO in controls and PA-treated cells, but the induced levels did not affect viability. ETV5 knockdown further resulted in significantly increased cellular ROS levels in response to PA and H2O2, but not in controls. PA treatment significantly induced p-AMPK after 24 h but not during 0-6 h. PA-induced p-AMPK levels were not changed by ETV5 knockout.

Conclusion NO plays no relevant role in PA-mediated lipotoxicity of INS-1E beta cells. ETV5 protects these cells by enabling the decomposition of increasing H2O2 concentrations during beta oxidation of palmitate. AMPK activation is not dependent on ETV5, but is associated with cellular ROS levels.

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Artikel online veröffentlicht:
02. Mai 2023