Astrocyte dysfunction is a primary factor in hepatic encephalopathy (HE) impairing neuronal activity under hyperammonemia. In particular the early events causing ammonia-induced toxicity to astrocytes are not well understood. Using established cellular HE models, we show that mitochondria rapidly undergo fragmentation in a reversible manner upon hyperammonemia. Further, within a timescale of minutes mitochondrial respiration and glycolysis were hampered which occurred in a pH-independent manner. Using metabolomics an accumulation of numerous amino acids, including branched chain amino acids and glucose was observed. Metabolomic tracking of 15 N-labeled ammonia showed rapid incorporation of 15 N into glutamate and glutamate-derived amino acids. Downregulating human GLUD2 , encoding mitochondrial glutamate dehydrogenase 2 (GDH2), inhibiting GDH2 activity by SIRT4 overexpression, and supplementing cells with glutamate or glutamine alleviated ammonia-induced inhibition of mitochondrial respiration. Metabolomic tracking of 13 C-glutamine showed that hyperammonemia can inhibit anaplerosis of TCA-cycle intermediates. Contrary to its classical anaplerotic role, we show that under hyperammonemia GDH2 rather catalyzes the removal of ammonia by reductive amination of α-ketoglutarate which efficiently and rapidly inhibits the TCA-cycle. Overall, we propose a critical GDH2-dependent mechanism in HE models that on the one hand helps to remove ammonia but on the other hand impairs energy metabolism in mitochondria rapidly.
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