Fig 1: Expression of key enzymes of the Krebs cycle by Western blotting in PC12 treated with corticosterone. (A) Expression levels of SUCLG2, ACO2, MDH1, CS, and IDH; (B-F) quantitative analysis of Western blotting results. The levels of Krebs cycle enzymes were significantly increased in KI vs. KT group. The experiments were repeated three times. The results are expressed as mean ± SD. *p < 0.05 vs. NC group. #p < 0.05 vs. KT group. GAPDH was used as the internal control. NC: No treatment control group; KT: Ketamine treatment; KI: Ketamine treatment with the inhibition of NF-?B; KO: Ketamine treatment with the overexpression of NF-?B. SUCLG2: Succinate-CoA ligase GDP-forming beta subunit; ACO2: Aconitase 2; MDH1: Malate dehydrogenase 1; CS: Citrate synthase; IDH: Isocitrate dehydrogenase; NF-?B: Nuclear factor kappa-light-chain-enhancer of activated B cells; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.
Fig 2: Cells Growing in Detached Conditions Have Increased Reductive Glutamine Metabolism(A and B) Isotopomer distribution of (A) citrate and (B) malate and fumarate in attached and detached 293T cells cultured with 13C5-glutamine for 4 h.(C) Levels and isotopomer distribution of 2-hydroxyglutarate in attached and detached cells cultured in the same conditions as (A). Peak area levels are normalized to cell number.(D) Representative GC-MS chromatogram showing the levels of L-and D-2-hydroxyglutarate in 293T cells cultured in detached and attached conditions.(E) 2-HG levels in 293T detached cells after siRNA knockdown of MDH1/2 and LDHA. Peak area levels are normalized to cell number.(F) Schematic representation of reductive glutamine metabolism in detached cells generating malate, 2-HG, and NAD+.(A, B, C, and E) Data are presented as ± SD of triplicate wells of representative experiments.
Fig 3: Mitochondrial Dysfunction Is Linked with Cell Migration(A) Enrichment p values (-log10) of gene ontology (GO) biological processes involved in cell migration and cytoskeleton remodeling as obtained with measurements of protein abundance by proteomics. Red dashed line indicates false discovery rate (FDR) = 0.05.(B and D) Migration speed of mT7, mT45, and mT80 cells (B) or shMDH1 mT80 cells (D) measured by wound healing assay.(C and E) Values of JATP consumption due to cytoskeleton remodeling based on calculations from OCR and ECAR data upon treatment with 1 µM nocodazole in mT7, mT45, and mT80 cells (C) or mT80 shMDH1 cells (E).(F) Immunofluorescence images of mT7, mT45, and mT80 cells stained with DAPI (blue), phalloidin (green), or antibody against MDH1 (red). White arrows indicate areas of co-localization between MDH1 and actin in mT80 cells.(G) Quantification of co-localization between MDH1 and phalloidin (actin). Data were obtained from 20–30 ROIs per condition.(B–E) Data are mean ± SEM from three to four independent cultures and were normalized on mean values of each experiment.*p = 0.05 and ***p = 0.001, ANOVA (B, C, and G) or Dunnett’s test (D).
Fig 4: Aspartate Transamination Supports Flux through MDH1 and Generation of Malate(A) Schematic representation of MAS and labeling patterns originating from (U)-13C-aspartate.(B and C) Proportion of total pool of malate m+4 (B) and fumarate m+4 (C) in mT7, mT45, and mT80 cells grown in the presence of U-13C-aspartate upon treatment with vehicle control or 0.5 µM rotenone.(D) Malate m+4 levels originating from (U)-13C-aspartate in mT80 cells upon silencing of GOT1. Data are normalized to intracellular levels of aspartate m+4 and are mean ± SD from one independent experiment.(E and F) Cell growth of mT7, mT45, and mT80 cells grown in 25 mM galactose and supplemented with 5 mM aspartate (E) upon treatment with 2 mM of the transaminase inhibitor aminooxyacetate (F). Data are normalized on cell growth of vehicle control (E) or on cell growth in the presence of aspartate only (F).(G) Cell growth of mT80 cells grown in 25 mM galactose and supplemented with 5 mM aspartate upon silencing of GOT1. Data are normalized to the cell growth rate of vehicle control.(H and I) Total levels of NAD+/NADH in mT7, mT45, and mT80 cells (H) or shMDH1 mT80 cells (I) upon supplementation with 5 mM aspartate. Data are normalized on vehicle control.(J and K) Secretion of lactate of mT7, mT45, and mT80 cells (J) or shMDH1 mT80 cells (K) upon supplementation with 5 mM aspartate. Data are normalized on vehicle control.(B, C, and E–K) Data are mean ± SEM from at least three independent cultures.*p = 0.05, **p = 0.01, ***p = 0.001, two-sided t test; n.s., not significant (B, C, E, and G–K). ***p = 0.001, one-way ANOVA (F).
Fig 5: Reductive Glutamine Carboxylation Regulates NAD Redox Balance and Supports Glycolysis in Response to Mitochondrial DysfunctionReduced turnover of NADH by mitochondria leads to impairment of the MAS and increase of cytosolic NADH. This in turn induces reductive carboxylation of glutamine, providing carbons for NADH-coupled MDH1, thus regulating NAD redox state and enhancing GAPDH activity. Increased glycolytic turnover supports ATP production in the cytosol, and this is associated with cell migration.
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