| Products/Services Used | Details | Operation |
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| Gene Synthesis | To visualize mitochondria, the construct pEGFP-Mito (Clontech Laboratories, Mountain View CA, USA) was used. Transfection was done using Effectene Transfection Reagent (Qiagen, Hilden, Germany) according to the manufacturer’s instructions 24 h before imaging. Knock-down of glutamate dehydrogenase 2 (GDH2, encoded by the GLUD2gene) using siRNAs (#NM_012084: 5’CUAACCUCUUCACGUGUAA’3 and 5’UUACACGUGAAGAGGUUAG‘3, Sigma-Aldrich, Taufkirchen, Germany) or transfection of GLUD2 plasmid/empty vector controls was done using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Carlsbad CA, USA), according to the manufacturer’s instructions using the reverse transfection protocol. Analysis occurred 48 h after transfection. For GLUD2 and empty vector plasmid transfections, as well as simultaneous transfection of plasmids and GDH siRNA, transfection was done directly in Seahorse cell plates. Human GLUD2 cloned into pcDNA3.1(+), Clone ID: OHu18663, was obtained from GenScript USA Inc., Piscataway NJ, USA. As control vector the empty pcDNA3.1(+) vector (Invitrogen, Thermo Fisher Scientific, Carlsbad CA, USA) was used. | Get A Quote |
In hepatic encephalopathy (HE) astrocyte dysfunction is a primary factor impairing neuronal activity under hyperammonemia. We show that mitochondria in cellular HE models undergo rapid fragmentation under hyperammonemia in a reversible manner. Mitochondrial respiration and glycolysis were instantaneously hampered in a pH-independent manner. A metabolomics approach revealed a subsequent accumulation of numerous amino acids, including branched chain amino acids, and glucose. N15labeling of ammonia shows rapid incorporation of ammonia-derived nitrogen into glutamate and glutamate-derived amino acids. Downregulating human GLUD2, encoding mitochondrial glutamate dehydrogenase 2 (GDH2), inhibiting GDH2 activity by S... More
