Brain pyruvate and 2-oxoglutarate dehydrogenase complexes are mitochondrial targets of the CoA ester of the Refsum disease marker phytanic acid

Pyruvate and 2-oxoglutarate dehydrogenase complexes are strongly inhibited by phytanoyl-CoA (IC(50) approximately 10(-6)-10(-7) M). Palmitoyl-CoA is 10-fold less potent. Phytanic or palmitic acids have no inhibitory effect up to 0.3 mM. At the substrate saturation, the acyl-CoA's affect the first and second enzymatic components of the 2-oxoglutarate dehydrogenase complex, while the third component is inhibited only at a low saturation with its substrate dihydrolipoamide. Thus, key regulatory branch points of mitochondrial metabolism are targets of a cellular derivative of phytanic acid. Decreased activity of the complexes might therefore contribute to neurological symptoms upon accumulation of phytanic acid in Refsum diseas

Predictive markers for efficiency of the amino-acid deprivation therapies in cancer

Amino acid deprivation therapy (AADT) is a promising strategy for developing novel anticancer treatments, based on variations in metabolism of healthy and malignant cells. L-asparaginase was the first amino acid-degrading enzyme that received FDA approval for the treatment of acute lymphoblastic leukemia (ALL). Arginase and arginine deiminase were effective in clinical trials for the treatment of metastatic melanomas and hepatocellular carcinomas. Essential dependence of certain cancer cells on methionine explains the anticancer efficacy of methionine-g-lyase. Along with significant progress in identification of metabolic vulnerabilities of cancer cells, new amino acid-cleaving enzymes appear as promising agents for cancer treatment: lysine oxidase, tyrosine phenol-lyase, cysteinase, and phenylalanine ammonia-lyase. However, sensitivity of specific cancer cell types to these enzymes differs. Hence, search for prognostic and predictive markers for AADT and introduction of the markers into clinical practice are of great importance for translational medicine. As specific metabolic pathways in cancer cells are determined by the enzyme expression, some of these enzymes may define the sensitivity to AADT. This review considers the known predictors for efficiency of AADT, emphasizing the importance of knowledge on cancer-specific amino acid significance for such predictions

Inhibition of 2-Oxoglutarate Dehydrogenase in Potato Tuber Suggests the Enzyme Is Limiting for Respiration and Confirms Its Importance in Nitrogen Assimilation1[W][OA]

The 2-oxoglutarate dehydrogenase complex constitutes a mitochondrially localized tricarboxylic acid cycle multienzyme system responsible for the conversion of 2-oxoglutarate to succinyl-coenzyme A concomitant with NAD+ reduction. Although regulatory mechanisms of plant enzyme complexes have been characterized in vitro, little is known concerning their role in plant metabolism in situ. This issue has recently been addressed at the cellular level in nonplant systems via the use of specific phosphonate inhibitors of the enzyme. Here, we describe the application of these inhibitors for the functional analysis of the potato (Solanum tuberosum) tuber 2-oxoglutarate dehydrogenase complex. In vitro experiments revealed that succinyl phosphonate (SP) and a carboxy ethyl ester of SP are slow-binding inhibitors of the 2-oxoglutarate dehydrogenase complex, displaying greater inhibitory effects than a diethyl ester of SP, a phosphono ethyl ester of SP, or a triethyl ester of SP. Incubation of potato tuber slices with the inhibitors revealed that they were adequately taken up by the tissue and produced the anticipated effects on the in situ enzyme activity. In order to assess the metabolic consequences of the 2-oxoglutarate dehydrogenase complex inhibition, we evaluated the levels of a broad range of primary metabolites using an established gas chromatography-mass spectrometry method. We additionally analyzed the rate of respiration in both tuber discs and isolated mitochondria. Finally, we evaluated the metabolic fate of radiolabeled acetate, 2-oxoglutarate or glucose, and 13C-labeled pyruvate and glutamate following incubation of tuber discs in the presence or absence of either SP or the carboxy ethyl ester of SP. The data obtained are discussed in the context of the roles of the 2-oxoglutarate dehydrogenase complex in respiration and carbon-nitrogen interactions

Daytime Dependence of the Activity of the Rat Brain Pyruvate Dehydrogenase Corresponds to the Mitochondrial Sirtuin 3 Level and Acetylation of Brain Proteins, All Regulated by Thiamine Administration Decreasing Phosphorylation of PDHA Ser293

Coupling glycolysis and mitochondrial tricarboxylic acid cycle, pyruvate dehydrogenase (PDH) complex (PDHC) is highly responsive to cellular demands through multiple mechanisms, including PDH phosphorylation. PDHC also produces acetyl-CoA for protein acetylation involved in circadian regulation of metabolism. Thiamine (vitamin B1) diphosphate (ThDP) is known to activate PDH as both coenzyme and inhibitor of the PDH inactivating kinases. Molecular mechanisms integrating the function of thiamine-dependent PDHC into general redox metabolism, underlie physiological fitness of a cell or an organism. Here, we characterize the daytime- and thiamine-dependent changes in the rat brain PDHC function, expression and phosphorylation, assessing their impact on protein acetylation and metabolic regulation. Morning administration of thiamine significantly downregulates both the PDH phosphorylation at Ser293 and SIRT3 protein level, the effects not observed upon the evening administration. This action of thiamine nullifies the daytime-dependent changes in the brain PDHC activity and mitochondrial acetylation, inducing diurnal difference in the cytosolic acetylation and acetylation of total brain proteins. Screening the daytime dependence of central metabolic enzymes and proteins of thiol/disulfide metabolism reveals that thiamine also cancels daily changes in the malate dehydrogenase activity, opposite to those of the PDHC activity. Correlation analysis indicates that thiamine abrogates the strong positive correlation between the total acetylation of the brain proteins and PDHC function. Simultaneously, thiamine heightens interplay between the expression of PDHC components and total acetylation or SIRT2 protein level. These thiamine effects on the brain acetylation system change metabolic impact of acetylation. The changes are exemplified by the thiamine enhancement of the SIRT2 correlations with metabolic enzymes and proteins of thiol-disulfide metabolism. Thus, we show the daytime- and thiamine-dependent changes in the function and phosphorylation of brain PDHC, contributing to regulation of the brain acetylation system and redox metabolism. The daytime-dependent action of thiamine on PDHC and SIRT3 may be of therapeutic significance in correcting perturbed diurnal regulation

Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays

Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay

Increasing Inhibition of the Rat Brain 2-Oxoglutarate Dehydrogenase Decreases Glutathione Redox State, Elevating Anxiety and Perturbing Stress Adaptation

Specific inhibitors of mitochondrial 2-oxoglutarate dehydrogenase (OGDH) are administered to animals to model the downregulation of the enzyme as observed in neurodegenerative diseases. Comparison of the effects of succinyl phosphonate (SP, 0.02 mmol/kg) and its uncharged precursor, triethyl succinyl phosphonate (TESP, 0.02 and 0.1 mmol/kg) reveals a biphasic response of the rat brain metabolism and physiology to increasing perturbation of OGDH function. At the low (TE)SP dose, glutamate, NAD+, and the activities of dehydrogenases of 2-oxoglutarate and malate increase, followed by their decreases at the high TESP dose. The complementary changes, i.e., an initial decrease followed by growth, are demonstrated by activities of pyruvate dehydrogenase and glutamine synthetase, and levels of oxidized glutathione and citrulline. While most of these indicators return to control levels at the high TESP dose, OGDH activity decreases and oxidized glutathione increases, compared to their control values. The first phase of metabolic perturbations does not cause significant physiological changes, but in the second phase, the ECG parameters and behavior reveal decreased adaptability and increased anxiety. Thus, lower levels of OGDH inhibition are compensated by the rearranged metabolic network, while the increased levels induce a metabolic switch to a lower redox state of the brain, associated with elevated stress of the animals