Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Oncometabolite d -2-hydroxyglutarate impairs α-ketoglutarate dehydrogenase and contractile function in rodent heart

doi: 10.1073/pnas.1601650113

Figure Lengend Snippet: D2-HG impairs cardiac energy metabolism by inhibiting α-KGDH. (A) Protocol for the isolated working rat heart with (0.5 mM or 1.0 mM) or without D2-HG. (B) LC-MS analysis of D2-HG concentration in hearts perfused with (0.5 mM or 1.0 mM) or without D2-HG (n = 3). (C and D) Hydraulic power (C) and glucose oxidation rate (D) at near-physiologic (100 cmH2O, 45–55 min) and increased workload (140 cmH2O, acute stimulation 55–58 min, prolonged stimulation 65–75 min). (E) Chemical structures of α-KG and D2-HG. (F) DARTs blotting showing D2-HG as a substrate of α-KGDH and ATP5B. Susceptibility of both α-KGDH and ATP5B to pronase digestion is increased in the presence of D2-HG. (G–I) Effect of D2-HG on α-KGDH activity (G), H2O2 production rate (H), and catalase activity (I) in mitochondria isolated from hearts perfused with or without D2-HG. (J) MMP assessed by 3,3′-dipropylthiadicarbocyanine iodide (DiSC35) staining and corrected by carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) of mitochondria isolated from hearts perfused with or without D2-HG. n = 3 rats per group. All data shown are mean ± SEM. Statistical analyses were performed with Kruskal–Wallis test, ANOVA, and Student’s t test. *P < 0.05; **P < 0.01; NS, not significant.

Article Snippet: /article/body/sec/ Simulations were performed using the mathematical model of mammalian cardiac metabolism CardioNet ( 14 ).

Techniques: Isolation, Liquid Chromatography with Mass Spectroscopy, Concentration Assay, Activity Assay, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Oncometabolite d -2-hydroxyglutarate impairs α-ketoglutarate dehydrogenase and contractile function in rodent heart

doi: 10.1073/pnas.1601650113

Figure Lengend Snippet: D2-HG affects energy substrate metabolism in the isolated working rat heart. (A) Protocol for the isolated working rat heart with physiologic concentrations of glucose (5 mM) and oleate (0.4 mM) in the presence or absence of D2-HG (1.0 mM). (B) LC-MS analysis of tissue D2-HG content in perfused rat hearts freeze-clamped at the end of the protocol. (C) Hydraulic power at near-physiologic (100 cmH2O, 45–55 min) and increased workload (140 cmH2O, acute stimulation 55–58 min, prolonged stimulation 65–75 min) in rat hearts perfused with or without D2-HG. (D) Measurement of glucose oxidation and oleate oxidation rates in isolated working rat hearts with or without D2-HG. (E) Analysis of glucose uptake and lactate release. (F) Effect of D2-HG on α-KGDH activity in mitochondria isolated from hearts perfused with or without D2-HG at physiologic concentrations of glucose and oleate. In A–F, n = 4 rats per group. Data are mean ± SEM *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant (Kruskal–Wallis test for perfusion data analysis, ANOVA and Student’s t test for pairwise comparisons).

Article Snippet: /article/body/sec/ Simulations were performed using the mathematical model of mammalian cardiac metabolism CardioNet ( 14 ).

Techniques: Isolation, Liquid Chromatography with Mass Spectroscopy, Activity Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Oncometabolite d -2-hydroxyglutarate impairs α-ketoglutarate dehydrogenase and contractile function in rodent heart

doi: 10.1073/pnas.1601650113

Figure Lengend Snippet: Flux rate analysis reveals dysregulation of cardiac energy substrate metabolism with increased D2-HG supply. Schematic of in silico flux rate analysis for glucose and D2-HG metabolism in ex vivo working heart perfusions. Colors indicate flux changes in the presence of D2-HG compared with control conditions. Ac-CoA, acetyl-CoA; Asp, aspartate; Fum, fumarate; Glut, glutamate; Homocys, homocysteine; Mal, malate; OAA, oxaloacetate; SAM, S-adenosylmethionine; Succ, succinate; Succ-CoA, succinyl-CoA; Tryp, tryptophane; and 5,10-Met-THF, 5,10-methenyl-tetrahydrofolate.

Article Snippet: /article/body/sec/ Simulations were performed using the mathematical model of mammalian cardiac metabolism CardioNet ( 14 ).

Techniques: In Silico, Ex Vivo, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Oncometabolite d -2-hydroxyglutarate impairs α-ketoglutarate dehydrogenase and contractile function in rodent heart

doi: 10.1073/pnas.1601650113

Figure Lengend Snippet: Identification of metabolic flux rate changes promoted by D2-HG. (A and B) Experimental measurements including uptake and release rates were input into a flux balance analysis using the metabolic model CardioNet (Table S1). Metabolic differences between the flux distributions in cardiac metabolism with or without D2-HG supply were analyzed to identify significant changes. Colors indicate calculated P values for each metabolic reaction in simulations without D2-HG supply compared with simulations with D2-HG supply. Metabolic reactions are shown according to their subcellular localization in the cytosol (A) or mitochondria (B) and clustered according to their association metabolic pathways. The minus logarithms of the P values are presented.

Article Snippet: /article/body/sec/ Simulations were performed using the mathematical model of mammalian cardiac metabolism CardioNet ( 14 ).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Oncometabolite d -2-hydroxyglutarate impairs α-ketoglutarate dehydrogenase and contractile function in rodent heart

doi: 10.1073/pnas.1601650113

Figure Lengend Snippet: Effect of D2-HG on skeletal muscle weight and cardiac metabolism. (A) Weight of left and right gastrocnemius muscles in mice after 32 d of D2-HG injection. (B) LC-MS analysis of D2-HG concentration in serum and heart tissue from mice treated with or without D2-HG. (C–E) Effect of D2-HG on α-KGDH activity (C), H2O2 production rate (D), and catalase activity (E) in isolated mitochondria from mice injected with D2-HG (n = 10). (F) Analysis of α-KG and succinate concentrations and [α-KG]:[succinate] ratio in perfused hearts freeze-clamped at the end of the protocol. (G) Analysis of HAT activities in nuclear fractions from heart tissue from mice injected with D2-HG. In A–G, n = 10 mice per group. Data expressed as mean ± SEM. Student’s t test. *P < 0.05, **P < 0.01; NS, not significant.

Article Snippet: /article/body/sec/ Simulations were performed using the mathematical model of mammalian cardiac metabolism CardioNet ( 14 ).

Techniques: Muscles, Injection, Liquid Chromatography with Mass Spectroscopy, Concentration Assay, Activity Assay, Isolation