Transcriptional and metabolic profiling of sulfur starvation response in two monocots.

BACKGROUND: Sulfur (S) is a mineral nutrient essential for plant growth and development, which is incorporated into diverse molecules fundamental for primary and secondary metabolism, plant defense, signaling, and maintaining cellular homeostasis. Although, S starvation response is well documented in the dicot model Arabidopsis thaliana, it is not clear if the same transcriptional networks control the response also in the monocots. RESULTS: We performed series of physiological, expression, and metabolite analyses in two model monocot species, one representing the C3 plants, Oryza sativa cv. kitaake, and second representing the C4 plants, Setaria viridis. Our comprehensive transcriptomic analysis revealed twice as many differentially expressed genes (DEGs) in S. viridis than in O. sativa under S-deficiency, consistent with a greater loss of sulfur and S-containing metabolites under these conditions. Surprisingly, most of the DEGs and enriched gene ontology terms were species-specific, with an intersect of only 58 common DEGs. The transcriptional networks were different in roots and shoots of both species, in particular no genes were down-regulated by S-deficiency in the roots of both species. CONCLUSIONS: Our analysis shows that S-deficiency seems to have different physiological consequences in the two monocot species and their nutrient homeostasis might be under distinct control mechanisms.

Sugar transporters spatially organize microbiota colonization along the longitudinal root axis of Arabidopsis.

Plant roots are functionally heterogeneous in cellular architecture, transcriptome profile, metabolic state, and microbial immunity. We hypothesized that axial differentiation may also impact spatial colonization by root microbiota along the root axis. We developed two growth systems, ArtSoil and CD-Rhizotron, to grow and then dissect Arabidopsis thaliana roots into three segments. We demonstrate that distinct endospheric and rhizosphere bacterial communities colonize the segments, supporting the hypothesis of microbiota differentiation along the axis. Root metabolite profiling of each segment reveals differential metabolite enrichment and specificity. Bioinformatic analyses and GUS histochemistry indicate microbe-induced accumulation of SWEET2, 4, and 12 sugar uniporters. Profiling of root segments from sweet mutants shows altered spatial metabolic profiles and reorganization of endospheric root microbiota. This work reveals the interdependency between root metabolites and microbial colonization and the contribution of SWEETs to spatial diversity and stability of microbial ecosystem.

Characterization of multiple lysophosphatidic acid acyltransferases in the plant pathogen Xanthomonas campestris.

Phosphatidic acid (PA) is the precursor of most phospholipids like phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. In bacteria, its biosynthesis begins with the acylation of glycerol-3-phosphate to lysophosphatidic acid (LPA), which is further acylated to PA by the PlsC enzyme. Some bacteria, like the plant pathogen Xanthomonas campestris, use a similar pathway to acylate lysophosphatidylcholine to phosphatidylcholine (PC). Previous studies assigned two acyltransferases to PC formation. Here, we set out to study their activity and found a second much more prominent function of these enzymes in LPA to PA conversion. This PlsC-like activity was supported by the functional complementation of a temperature-sensitive plsC-deficient Escherichia coli strain. Biocomputational analysis revealed two further PlsC homologs in X. campestris. The cellular levels of the four PlsC-like proteins varied with respect to growth phase and growth temperature. To address the question whether these enzymes have redundant or specific functions, we purified two recombinant, detergent-solubilized enzymes in their active form, which enabled the first direct biochemical comparison of PlsC isoenzymes from the same organism. Overlapping but not identical acyl acceptor and acyl donor preferences suggest redundant and specialized functions of the X. campestris PlsC enzymes. The altered fatty acid composition in plsC mutant strains further supports the functional differentiation of these enzymes.

The role of metabolomics in informing strategies for improving photosynthesis.

Photosynthesis plays a vital role in acclimating to and mitigating climate change, providing food and energy security for a population that is constantly growing, and achieving an economy with zero carbon emissions. A thorough comprehension of the dynamics of photosynthesis, including its molecular regulatory network and limitations, is essential for utilizing it as a tool to boost plant growth, enhance crop yields, and support the production of plant biomass for carbon storage. Photorespiration constrains photosynthetic efficiency and contributes significantly to carbon loss. Therefore, modulating or circumventing photorespiration presents opportunities to enhance photosynthetic efficiency. Over the past eight decades, substantial progress has been made in elucidating the molecular basis of photosynthesis, photorespiration, and the key regulatory mechanisms involved, beginning with the discovery of the canonical Calvin-Benson-Bassham cycle. Advanced chromatographic and mass spectrometric technologies have allowed a comprehensive analysis of the metabolite patterns associated with photosynthesis, contributing to a deeper understanding of its regulation. In this review, we summarize the results of metabolomics studies that shed light on the molecular intricacies of photosynthetic metabolism. We also discuss the methodological requirements essential for effective analysis of photosynthetic metabolism, highlighting the value of this technology in supporting strategies aimed at enhancing photosynthesis.

Sphingosine-1-phosphate suppresses GLUT activity through PP2A and counteracts hyperglycemia in diabetic red blood cells.

Red blood cells (RBC) are the major carriers of sphingosine-1-phosphate (S1P) in blood. Here we show that variations in RBC S1P content achieved by altering S1P synthesis and transport by genetic and pharmacological means regulate glucose uptake and metabolic flux. This is due to S1P-mediated activation of the catalytic protein phosphatase 2 (PP2A) subunit leading to reduction of cell-surface glucose transporters (GLUTs). The mechanism dynamically responds to metabolic cues from the environment by increasing S1P synthesis, enhancing PP2A activity, reducing GLUT phosphorylation and localization, and diminishing glucose uptake in RBC from diabetic mice and humans. Functionally, it protects RBC against lipid peroxidation in hyperglycemia and diabetes by activating the pentose phosphate pathway. Proof of concept is provided by the resistance of mice lacking the S1P exporter MFSD2B to diabetes-induced HbA1c elevation and thiobarbituric acid reactive substances (TBARS) generation in diabetic RBC. This mechanism responds to pharmacological S1P analogues such as fingolimod and may be functional in other insulin-independent tissues making it a promising therapeutic target.

Brassicaceae display variation in efficiency of photorespiratory carbon-recapturing mechanisms.

Carbon-concentrating mechanisms enhance the carboxylase efficiency of Rubisco by providing supra-atmospheric concentrations of CO2 in its surroundings. Beside the C4 photosynthesis pathway, carbon concentration can also be achieved by the photorespiratory glycine shuttle which requires fewer and less complex modifications. Plants displaying CO2 compensation points between 10 ppm and 40 ppm are often considered to utilize such a photorespiratory shuttle and are termed 'C3-C4 intermediates'. In the present study, we perform a physiological, biochemical, and anatomical survey of a large number of Brassicaceae species to better understand the C3-C4 intermediate phenotype, including its basic components and its plasticity. Our phylogenetic analysis suggested that C3-C4 metabolism evolved up to five times independently in the Brassicaceae. The efficiency of the pathway showed considerable variation. Centripetal accumulation of organelles in the bundle sheath was consistently observed in all C3-C4-classified taxa, indicating a crucial role for anatomical features in CO2-concentrating pathways. Leaf metabolite patterns were strongly influenced by the individual species, but accumulation of photorespiratory shuttle metabolites glycine and serine was generally observed. Analysis of phosphoenolpyruvate carboxylase activities suggested that C4-like shuttles have not evolved in the investigated Brassicaceae. Convergent evolution of the photorespiratory shuttle indicates that it represents a distinct photosynthesis type that is beneficial in some environments.

Pancreatic islet protection at the expense of secretory function involves serine-linked mitochondrial one-carbon metabolism.

Type 2 diabetes is characterized by insulin hypersecretion followed by reduced glucose-stimulated insulin secretion (GSIS). Here we show that acute stimulation of pancreatic islets with the insulin secretagogue dextrorphan (DXO) or glibenclamide enhances GSIS, whereas chronic treatment with high concentrations of these drugs reduce GSIS but protect islets from cell death. Bulk RNA sequencing of islets shows increased expression of genes for serine-linked mitochondrial one-carbon metabolism (OCM) after chronic, but not acute, stimulation. In chronically stimulated islets, more glucose is metabolized to serine than to citrate, and the mitochondrial ATP/ADP ratio decreases, whereas the NADPH/NADP+ ratio increases. Activating transcription factor-4 (Atf4) is required and sufficient to activate serine-linked mitochondrial OCM genes in islets, with gain- and loss-of-function experiments showing that Atf4 reduces GSIS and is required, but not sufficient, for full DXO-mediated islet protection. In sum, we identify a reversible metabolic pathway that provides islet protection at the expense of secretory function.

A systematic overexpression approach reveals native targets to increase squalene production in Synechocystis sp. PCC 6803.

Cyanobacteria are a promising platform for the production of the triterpene squalene (C30), a precursor for all plant and animal sterols, and a highly attractive intermediate towards triterpenoids, a large group of secondary plant metabolites. Synechocystis sp. PCC 6803 natively produces squalene from CO2 through the MEP pathway. Based on the predictions of a constraint-based metabolic model, we took a systematic overexpression approach to quantify native Synechocystis gene's impact on squalene production in a squalene-hopene cyclase gene knock-out strain (Δshc). Our in silico analysis revealed an increased flux through the Calvin-Benson-Bassham cycle in the Δshc mutant compared to the wildtype, including the pentose phosphate pathway, as well as lower glycolysis, while the tricarboxylic acid cycle predicted to be downregulated. Further, all enzymes of the MEP pathway and terpenoid synthesis, as well as enzymes from the central carbon metabolism, Gap2, Tpi and PyrK, were predicted to positively contribute to squalene production upon their overexpression. Each identified target gene was integrated into the genome of Synechocystis Δshc under the control of the rhamnose-inducible promoter Prha. Squalene production was increased in an inducer concentration dependent manner through the overexpression of most predicted genes, which are genes of the MEP pathway, ispH, ispE, and idi, leading to the greatest improvements. Moreover, we were able to overexpress the native squalene synthase gene (sqs) in Synechocystis Δshc, which reached the highest production titer of 13.72 mg l-1 reported for squalene in Synechocystis sp. PCC 6803 so far, thereby providing a promising and sustainable platform for triterpene production.

Carbon monoxide exposure activates ULK1 via AMPK phosphorylation in murine embryonic fibroblasts.

Carbon monoxide (CO) is endogenously produced upon degradation of heme by heme oxygenases (HOs) and is suggested to act as a gaseous signaling molecule. The expression of HO-1 is triggered by the Nrf2-Keap1 signaling pathway which responds to exogenous stress signals and dietary constituents such as flavonoids and glucosinolates or reactive metabolic intermediates like 4-hydroxynonenal. Endogenous CO affects energy metabolism, regulates the utilization of glucose and addresses CYP450 enzymes. Using the CO releasing molecule-401 (CORM-401), we studied the effect of endogenous CO on ATP synthesis, AMP-signaling and activation of the AMPK pathway in cell culture. Upon exposure of cells to CORM-401, the mitochondrial ATP production rate was significantly decreased (P=0.007) to about 50%, while glycolytic ATP synthesis was unchanged (P=0.489). Total ATP levels were less affected as determined by mass spectrometry. Instead, levels of ADP and AMP were elevated following CORM-401 exposure by about two- (P=0.022) and four-fold (P=0.012) compared to control, respectively. Increased concentrations of AMP activate AMPK which was demonstrated by a 10 to 15-fold increased phosphorylation of Thr172 of the α-subunit of AMPK (P=0.025). A downstream target of AMPK is the kinase ULK1 which triggers autophagic and mitophagic processes. Activation of ULK1 after CO exposure was proven by a 3 to 5-fold elevated phosphorylation of ULK1 at Ser555 (P=0.004). The present data suggest that production of endogenous CO leads to increasing amounts of AMP which mediates AMPK-dependent downstream effects and likely triggers autophagic processes. Since dietary constituents and their metabolites induce the expression of the CO producing enzyme HO-1, CO signaling may also be involved in the cellular response to nutritional factors.

WNT/ß-Catenin-Mediated Resistance to Glucose Deprivation in Glioblastoma Stem-like Cells.

Isocitrate dehydrogenase (IDH)-wildtype glioblastoma is the most common primary malignant brain tumor. It is associated with a particularly poor prognosis, as reflected by an overall median survival of only 15 months in patients who undergo a supramarginal surgical reduction of the tumor mass followed by combined chemoradiotherapy. The highly malignant nature of IDH-wildtype glioblastoma is thought to be driven by glioblastoma stem-like cells (GSCs) that harbor the ability of self-renewal, survival, and adaptability to challenging environmental conditions. The wingless (WNT) signaling pathway is a phylogenetically highly conserved stemness pathway, which promotes metabolic plasticity and adaptation to a nutrient-limited tumor microenvironment. To unravel the reciprocal regulation of the WNT pathway and the nutrient-limited microenvironment, glioblastoma cancer stem-like cells were cultured in a medium with either standard or reduced glucose concentrations for various time points (24, 48, and 72 h). Glucose depletion reduced cell viability and facilitated the survival of a small population of starvation-resistant tumor cells. The surviving cells demonstrated increased clonogenic and invasive properties as well as enhanced chemosensitivity to pharmacological inhibitors of the WNT pathway (LGK974, berberine). Glucose depletion partially led to the upregulation of WNT target genes such as CTNNB1, ZEB1, and AXIN2 at the mRNA and corresponding protein levels. LGK974 treatment alone or in combination with glucose depletion also altered the metabolite concentration in intracellular compartments, suggesting WNT-mediated metabolic regulation. Taken together, our findings suggest that WNT-mediated metabolic plasticity modulates the survival of GSCs under nutrient-restricted environmental conditions.

Improvement of prediction ability by integrating multi-omic datasets in barley.

BACKGROUND: Genomic prediction (GP) based on single nucleotide polymorphisms (SNP) has become a broadly used tool to increase the gain of selection in plant breeding. However, using predictors that are biologically closer to the phenotypes such as transcriptome and metabolome may increase the prediction ability in GP. The objectives of this study were to (i) assess the prediction ability for three yield-related phenotypic traits using different omic datasets as single predictors compared to a SNP array, where these omic datasets included different types of sequence variants (full-SV, deleterious-dSV, and tolerant-tSV), different types of transcriptome (expression presence/absence variation-ePAV, gene expression-GE, and transcript expression-TE) sampled from two tissues, leaf and seedling, and metabolites (M); (ii) investigate the improvement in prediction ability when combining multiple omic datasets information to predict phenotypic variation in barley breeding programs; (iii) explore the predictive performance when using SV, GE, and ePAV from simulated 3'end mRNA sequencing of different lengths as predictors. RESULTS: The prediction ability from genomic best linear unbiased prediction (GBLUP) for the three traits using dSV information was higher than when using tSV, all SV information, or the SNP array. Any predictors from the transcriptome (GE, TE, as well as ePAV) and metabolome provided higher prediction abilities compared to the SNP array and SV on average across the three traits. In addition, some (di)-similarity existed between different omic datasets, and therefore provided complementary biological perspectives to phenotypic variation. Optimal combining the information of dSV, TE, ePAV, as well as metabolites into GP models could improve the prediction ability over that of the single predictors alone. CONCLUSIONS: The use of integrated omic datasets in GP model is highly recommended. Furthermore, we evaluated a cost-effective approach generating 3'end mRNA sequencing with transcriptome data extracted from seedling without losing prediction ability in comparison to the full-length mRNA sequencing, paving the path for the use of such prediction methods in commercial breeding programs.

Multiomics of synaptic junctions reveals altered lipid metabolism and signaling following environmental enrichment.

Membrane lipids and their metabolism have key functions in neurotransmission. Here we provide a quantitative lipid inventory of mouse and rat synaptic junctions. To this end, we developed a multiomics extraction and analysis workflow to probe the interplay of proteins and lipids in synaptic signal transduction from the same sample. Based on this workflow, we generate hypotheses about novel mechanisms underlying complex changes in synaptic connectivity elicited by environmental stimuli. As a proof of principle, this approach reveals that in mice exposed to an enriched environment, reduced endocannabinoid synthesis and signaling is linked to increased surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) in a subset of Cannabinoid-receptor 1 positive synapses. This mechanism regulates synaptic strength in an input-specific manner. Thus, we establish a compartment-specific multiomics workflow that is suitable to extract information from complex lipid and protein networks involved in synaptic function and plasticity.

Metabolic engineering of Synechocystis sp. PCC 6803 for the photoproduction of the sesquiterpene valencene.

Cyanobacteria are extremely adaptable, fast-growing, solar-powered cell factories that, like plants, are able to convert carbon dioxide into sugar and oxygen and thereby produce a large number of important compounds. Due to their unique phototrophy-associated physiological properties, i.e. naturally occurring isoprenoid metabolic pathway, they represent a highly promising platform for terpenoid biosynthesis. Here, we implemented a carefully devised engineering strategy to boost the biosynthesis of commercially attractive plant sequiterpenes, in particular valencene. Sesquiterpenes are a diverse group of bioactive metabolites, mainly produced in higher plants, but with often low concentrations and expensive downstream extraction. In this work we successfully demonstrate a multi-component engineering approach towards the photosynthetic production of valencene in the cyanobacterium Synechocystis sp. PCC 6803. First, we improved the flux towards valencene by markerless genomic deletions of shc and sqs. Secondly, we downregulated the formation of carotenoids, which are essential for viability of the cell, using CRISPRi on crtE. Finally, we intended to increase the spatial proximity of the two enzymes, ispA and CnVS, involved in valencene formation by creating an operon construct, as well as a fusion protein. Combining the most successful strategies resulted in a valencene production of 19 mg/g DCW in Synechocystis. In this work, we have devised a useful platform for future engineering steps.

Targeted Phosphoinositides Analysis Using High-Performance Ion Chromatography-Coupled Selected Reaction Monitoring Mass Spectrometry.

Phosphoinositides are minor components of cell membranes, but play crucial roles in numerous signal transduction pathways. To obtain quantitative measures of phosphoinositides, sensitive, accurate, and comprehensive methods are needed. Here, we present a quantitative targeted ion chromatography-mass spectrometry-based workflow that separates phosphoinositide isomers and increases the quantitative accuracy of measured phosphoinositides. Besides testing different analytical characteristics such as extraction and separation efficiency, the reproducibility of the developed workflow was also investigated. The workflow was verified in resting and stimulated human platelets, fat cells, and rat hippocampal brain tissue, where the LOD and LOQ for phosphoinositides were at 312.5 and 625 fmol, respectively. The robustness of the workflow is shown with different applications that confirms its suitability to analyze multiple less-abundant phosphoinositides.

A synthetic C4 shuttle via the ß-hydroxyaspartate cycle in C3 plants.

Plants depend on the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) for CO2 fixation. However, especially in C3 plants, photosynthetic yield is reduced by formation of 2-phosphoglycolate, a toxic oxygenation product of Rubisco, which needs to be recycled in a high-flux-demanding metabolic process called photorespiration. Canonical photorespiration dissipates energy and causes carbon and nitrogen losses. Reducing photorespiration through carbon-concentrating mechanisms, such as C4 photosynthesis, or bypassing photorespiration through metabolic engineering is expected to improve plant growth and yield. The ß-hydroxyaspartate cycle (BHAC) is a recently described microbial pathway that converts glyoxylate, a metabolite of plant photorespiration, into oxaloacetate in a highly efficient carbon-, nitrogen-, and energy-conserving manner. Here, we engineered a functional BHAC in plant peroxisomes to create a photorespiratory bypass that is independent of 3-phosphoglycerate regeneration or decarboxylation of photorespiratory precursors. While efficient oxaloacetate conversion in Arabidopsis thaliana still masks the full potential of the BHAC, nitrogen conservation and accumulation of signature C4 metabolites demonstrate the proof of principle, opening the door to engineering a photorespiration-dependent synthetic carbon-concentrating mechanism in C3 plants.

New Insights Into the Evolution of C4 Photosynthesis Offered by the Tarenaya Cluster of Cleomaceae.

Cleomaceae is closely related to Brassicaceae and includes C3, C3-C4, and C4 species. Thus, this family represents an interesting system for studying the evolution of the carbon concentrating mechanism. However, inadequate genetic information on Cleomaceae limits their research applications. Here, we characterized 22 Cleomaceae accessions [3 genera (Cleoserrata, Gynandropsis, and Tarenaya) and 11 species] in terms of genome size; molecular phylogeny; as well as anatomical, biochemical, and photosynthetic traits. We clustered the species into seven groups based on genome size. Interestingly, despite clear differences in genome size (2C, ranging from 0.55 to 1.3 pg) in Tarenaya spp., this variation was not consistent with phylogenetic grouping based on the internal transcribed spacer (ITS) marker, suggesting the occurrence of multiple polyploidy events within this genus. Moreover, only G. gynandra, which possesses a large nuclear genome, exhibited the C4 metabolism. Among the C3-like species, we observed intra- and interspecific variation in nuclear genome size as well as in biochemical, physiological, and anatomical traits. Furthermore, the C3-like species had increased venation density and bundle sheath cell size, compared to C4 species, which likely predisposed the former lineages to C4 photosynthesis. Accordingly, our findings demonstrate the potential of Cleomaceae, mainly members of Tarenaya, in offering novel insights into the evolution of C4 photosynthesis.

Crystal Structure and Pathophysiological Role of the Pneumococcal Nucleoside-binding Protein PnrA.

Nucleotides are important for RNA and DNA synthesis and, despite a de novo synthesis by bacteria, uptake systems are crucial. Streptococcus pneumoniae, a facultative human pathogen, produces a surface-exposed nucleoside-binding protein, PnrA, as part of an ABC transporter system. Here we demonstrate the binding affinity of PnrA to nucleosides adenosine, guanosine, cytidine, thymidine and uridine by microscale thermophoresis and indicate the consumption of adenosine and guanosine by 1H NMR spectroscopy. In a series of five crystal structures we revealed the PnrA structure and provide insights into how PnrA can bind purine and pyrimidine ribonucleosides but with preference for purine ribonucleosides. Crystal structures of PnrA:nucleoside complexes unveil a clear pattern of interactions in which both the N- and C- domains of PnrA contribute. The ribose moiety is strongly recognized through a conserved network of H-bond interactions, while plasticity in loop 27-36 is essential to bind purine- or pyrimidine-based nucleosides. Further, we deciphered the role of PnrA in pneumococcal fitness in infection experiments. Phagocytosis experiments did not show a clear difference in phagocytosis between PnrA-deficient and wild-type pneumococci. In the acute pneumonia infection model the deficiency of PnrA attenuated moderately virulence of the mutant, which is indicated by a delay in the development of severe lung infections. Importantly, we confirmed the loss of fitness in co-infections, where the wild-type out-competed the pnrA-mutant. In conclusion, we present the PnrA structure in complex with individual nucleosides and show that the consumption of adenosine and guanosine under infection conditions is required for virulence.

An intact gut microbiome protects genetically predisposed mice against leukemia.

The majority of childhood leukemias are precursor B-cell acute lymphoblastic leukemias (pB-ALLs) caused by a combination of prenatal genetic predispositions and oncogenic events occurring after birth. Although genetic predispositions are frequent in children (>1% to 5%), fewer than 1% of genetically predisposed carriers will develop pB-ALL. Although infectious stimuli are believed to play a major role in leukemogenesis, the critical determinants are not well defined. Here, by using murine models of pB-ALL, we show that microbiome disturbances incurred by antibiotic treatment early in life were sufficient to induce leukemia in genetically predisposed mice, even in the absence of infectious stimuli and independent of T cells. By using V4 and full-length 16S ribosomal RNA sequencing of a series of fecal samples, we found that genetic predisposition to pB-ALL (Pax5 heterozygosity or ETV6-RUNX1 fusion) shaped a distinct gut microbiome. Machine learning accurately (96.8%) predicted genetic predisposition using 40 of 3983 amplicon sequence variants as proxies for bacterial species. Transplantation of either wild-type (WT) or Pax5+/- hematopoietic bone marrow cells into WT recipient mice revealed that the microbiome is shaped and determined in a donor genotype-specific manner. Gas chromatography-mass spectrometry (GC-MS) analyses of sera from WT and Pax5+/- mice demonstrated the presence of a genotype-specific distinct metabolomic profile. Taken together, our data indicate that it is a lack of commensal microbiota rather than the presence of specific bacteria that promotes leukemia in genetically predisposed mice. Future large-scale longitudinal studies are required to determine whether targeted microbiome modification in children predisposed to pB-ALL could become a successful prevention strategy.

Ammonia inhibits energy metabolism in astrocytes in a rapid and glutamate dehydrogenase 2-dependent manner.

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, in our analyses, within a timescale of minutes, mitochondrial respiration and glycolysis were hampered, which occurred in a pH-independent manner. Using metabolomics, an accumulation of glucose and numerous amino acids, including branched chain amino acids, was observed. Metabolomic tracking of 15N-labeled ammonia showed rapid incorporation of 15N 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 13C-glutamine showed that hyperammonemia can inhibit anaplerosis of tricarboxylic acid (TCA) cycle intermediates. Contrary to its classical anaplerotic role, we show that, under hyperammonemia, GDH2 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 helps to remove ammonia, but also impairs energy metabolism in mitochondria rapidly.