Figure 2.

High salt inhibits mitochondrial electron transport chain at complex II. A, Pictogram of the central carbon metabolism and ETC. Glucose-derived ¹³C-label is represented as red dots. Glutamine-derived ¹³C-label is represented as blue dots. Mitochondrial membrane potential is ψ. B through D, Bone marrow–derived macrophages (BMDMs) treated for 3 h (B and D) or 24 h (C) with lipopolysaccharide (LPS) or interleukin (IL) 4+IL13 under normal salt (NS) or high salt (HS) conditions. B and C, ¹³C-glutamine-derived ¹³C-fumarate to ¹³C-succinate ratio as surrogate for succinate dehydrogenase/complex II activity (n=5). Data are depicted as box and whisker with minimum to maximum. Significance was analyzed by unpaired 2-tailed t test. D, Relative (deproteinized) FAD content (n=4). Data are depicted as box and whisker with minimum to maximum. Significance was analyzed by unpaired 2-tailed t test. E, Relative (deproteinized) FAD content in freshly isolated monocytes (n=6 donors), cultivated for 3 hours under NS or HS conditions. Data are depicted in relation to the donor-paired NS control. Significance was analyzed by Wilcoxon matched-pairs signed-rank 2-tailed test. F, Relative complex II activity after purification of complex II from BMDMs under increasing concentrations of additional NaCl (0 mM to 4 mM) (n=3 or 4). Data are depicted as mean in relation to the NS control. Significance was analyzed by 1-way ANOVA with Tukey’s post hoc test. G, Relative complex II+III activity in bovine heart mitochondria under increasing concentrations of additional NaCl (0.0625 mM to 64 mM) or antimycin A (AA, 0.3438 nM to 352 nM) (n=3 per concentration, respectively). H, Relative complex IV activity in bovine heart mitochondria under increasing concentrations of additional NaCl (0.0625 mM to 64 mM) or potassium cyanide (KCN, 0.01 µM to 100 µM) (n=3 for NaCl and n=1 for KCN). Data are depicted as mean±SEM in relation to the NS control for G and H. I, Relative complex I activity after purification of complex I from BMDMs under increasing concentrations of additional NaCl (0 mM to 4 mM) (n=3). Data are depicted as mean in relation to the NS control. Significance was analyzed by 1-way ANOVA with Tukey’s post hoc test. J, NAD⁺ to NADH ratio as surrogate for complex I activity in BMDMs treated for 3 hours with LPS or IL4+IL13 under NS, HS, or NS+rotenone (10 µM) treatment (n=6). Data are depicted as box and whisker with minimum to maximum. Significance was analyzed by 1-way ANOVA with Tukey’s post hoc test. 2OG indicates 2-Oxoglutarate; ATP, Adenosine Triphosphate; Cit, Citrate; ETC, electron transport chain; FAD, Flavin Adenine Dinucleotide; Fh, Fumarate hydratase; Fum, Fumarate; Glc, Glucose; Gln, Glutamine; Glu, Glutamate; Glud1, Glutamate Dehydrogenase 1; H+, protons; Ita, Itaconate; Lac, Lactate; Ldha, Lactate Dehydrogenase A; Mal, Malate; NAD+, oxidized Nicotinamide Adenine Dinucleotide; NADH+H+, reduced Nicotinamide Adenine Dinucleotide; O2, oxygen; OAA, Oxaloacetate; Pcx, Pyruvate Carboxylase; Pdha, Pyruvate Dehydrogenase E1 Subunit Alpha 1; Pyr, Pyruvate; Sdha, Succinate Dehydrogenase complex, subunit A; and Suc, Succinate.