Ammonia inhibits energy metabolism in astrocytes in a rapid and GDH2-dependent manner

Abstract

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. N15 labeling 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 SIRT4 overexpression, and supplementing cells with glutamate or glutamine alleviated ammonia-induced inhibition of mitochondrial respiration. Thus, under hyperammonemic conditions, GDH2 catalyzes the removal of ammonia by reductive amination of α-ketoglutarate but at the same time inhibits the TCA-cycle by depleting α-ketoglutarate. Overall, we propose a mitochondria-dependent mechanism contributing to the early steps in the pathogenesis of HE where the interplay between energy metabolism and ammonia removal plays a pivotal role.

Subcellular Localization and Mitotic Interactome Analyses Identify SIRT4 as a Centrosomally Localized and Microtubule Associated Protein

“…HEK293 cell lines stably expressing SIRT4-eGFP from the pcDNA3.1 vector and mutants [enzymatically inactive SIRT4(H161Y)-eGFP and SIRT4(ΔN28)-eGFP lacking the N-terminal mitochondrial targeting signal] have been described [ 42 ] and cultured in media containing 800 µg/mL Geneticin/G418 (Genaxxon bioscience GmbH, Ulm, Germany) as permanent selection agent. HEK293 and HeLa cell lines stably expressing SIRT4-eGFP from the retroviral vector puc2CL12IPwo were generated as described elsewhere [ 43 ] and further enriched by fluorescence-activated cell sorting (FACS). Expression of SIRT4-eGFP fusion constructs was validated by immunoblotting and flow cytometry.…”

New Insight Into Mechanisms of Hepatic Encephalopathy: An Integrative Analysis Approach to Identify Molecular Markers and Therapeutic Targets

“…Glutamate toxicity, mitochondrial damage, depletion of cellular ATP stores, and decreased energy metabolism have been reported in the brain of animal models of HE 86 , 87 as well as ammonia-exposed astrocyte cultures. 88 - 91 Exposure of the primary culture of astrocytes to glutamate inhibits fatty acid beta-oxidation. 81 Glutamate toxicity and mitochondrial impairments may be responsible for ATP depletion and decreased brain energy metabolisms following HE due to the inhibition of astroglial fatty acid oxidation.…”

Subcellular localization and mitotic interactome analyses identify SIRT4 as a centrosomally localized and microtubule associated protein

“…HEK293 cell lines stably expressing SIRT4-eGFP from the pcDNA3.1 vector and mutants [enzymatically inactive SIRT4(H161Y)-eGFP and SIRT4(N28)-eGFP lacking the N-terminal mitochondrial targeting signal] have been described42 and cultured in media containing 800 µg/ml Geneticin/G418 (Genaxxon) as permanent selection agent. HEK293 and HeLa cell lines stably expressing SIRT4-eGFP from the retroviral vector puc2CL12IPwo were generated as described elsewhere74 and further enriched by fluorescence activated cell sorting (FACS). Expression of SIRT4-eGFP fusion constructs was validated by immunoblotting and flow cytometry.Cell proliferation kinetics.…”