A. V. Graf

On the role of the mitochondrial 2-oxoglutarate dehydrogenase complex in amino acid metabolism.

Mitochondria are tightly linked to cellular nutrient sensing, and provide not only energy, but also intermediates for the de novo synthesis of cellular compounds including amino acids. Mitochondrial metabolic enzymes as generators and/or targets of signals are therefore important players in the distribution of intermediates between catabolic and anabolic pathways. The highly regulated 2-oxoglutarate dehydrogenase complex (OGDHC) participates in glucose oxidation via the tricarboxylic acid cycle. It occupies an amphibolic branch point in the cycle, where the energy-producing reaction of the 2-oxoglutarate degradation competes with glutamate (Glu) synthesis via nitrogen incorporation into 2-oxoglutarate. To characterize the specific impact of the OGDHC inhibition on amino acid metabolism in both plant and animal mitochondria, a synthetic analog of 2-oxoglutarate, namely succinyl phosphonate (SP), was applied to living systems from different kingdoms, both in situ and in vivo. Using a high-throughput mass spectrometry-based approach, we showed that organisms possessing OGDHC respond to SP by significantly changing their amino acid pools. By contrast, cyanobacteria which lack OGDHC do not show perturbations in amino acids following SP treatment. Increases in Glu, 4-aminobutyrate and alanine represent the most universal change accompanying the 2-oxoglutarate accumulation upon OGDHC inhibition. Other amino acids were affected in a species-specific manner, suggesting specific metabolic rearrangements and substrate availability mediating secondary changes. Strong perturbation in the relative abundance of amino acids due to the OGDHC inhibition was accompanied by decreased protein content. Our results provide specific evidence of a considerable role of OGDHC in amino acid metabolism.

Up-regulation of 2-oxoglutarate dehydrogenase as a stress response

2-Oxoglutarate dehydrogenase multienzyme complex (OGDHC) operates at a metabolic cross-road, mediating Ca(2+)- and ADP-dependent signals in mitochondria. Here, we test our hypothesis that OGDHC plays a major role in the neurotransmitter metabolism and associated stress response. This possibility was assessed using succinyl phosphonate (SP), a highly specific and efficient in vivo inhibitor of OGDHC. Animals exposed to toxicants (SP, ethanol or MnCl(2)), trauma or acute hypoxia showed intrinsic up-regulation of OGDHC in brain and heart. The known mechanism of the SP action as OGDHC inhibitor pointed to the up-regulation triggered by the enzyme impairment. The animal behavior and skeletal muscle or heart performance were tested to correlate physiology with the OGDHC regulation and associated changes in the glutamate and cellular energy status. The SP-treated animals exhibited interdependent changes in the brain OGDHC activity, glutamate level and cardiac autonomic balance, suggesting the neurotransmitter role of glutamate to be involved in the changed heart performance. Energy insufficiency after OGDHC inhibition was detectable neither in animals up to 25 mg/kg SP, nor in cell culture during 24 h incubation with 0.1 mM SP. However, in animals subjected to acute ethanol intoxication SP did evoke energy deficit, decreasing muscular strength and locomotion and increasing the narcotic sleep duration. This correlated with the SP-induced decrease in NAD(P)H levels of the ethanol-exposed neurons. Thus, we show the existence of natural mechanisms to up-regulate mammalian OGDHC in response to stress, with both the glutamate neurotransmission and energy production potentially involved in the OGDHC impact on physiological performance. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.

Behavioral Impact of the Regulation of the Brain 2-Oxoglutarate Dehydrogenase Complex by Synthetic Phosphonate Analog of 2-Oxoglutarate: Implications into the Role of the Complex in Neurodegenerative Diseases

Decreased activity of the mitochondrial 2-oxoglutarate dehydrogenase complex (OGDHC) in brain accompanies neurodegenerative diseases. To reveal molecular mechanisms of this association, we treated rats with a specific inhibitor of OGDHC, succinyl phosphonate, or exposed them to hypoxic stress. In males treated with succinyl phosphonate and in pregnancy-sensitized females experiencing acute hypobaric hypoxia, we revealed upregulation of brain OGDHC (within 24 hours), with the activity increase presumably representing the compensatory response of brain to the OGDHC inhibition. This up-regulation of brain OGDHC was accompanied by an increase in exploratory activity and a decrease in anxiety of the experimental animals. Remarkably, the hypoxia-induced elevation of brain OGDHC and most of the associated behavioral changes were abrogated by succinyl phosphonate. The antagonistic action of hypoxia and succinyl phosphonate demonstrates potential therapeutic significance of the OGDHC regulation by the phosphonate analogs of 2-oxoglutarate.

Effect of hypoxia during early organogenesis on cardiac activity and noradrenergic regulation in the postnatal period

Cardiac activity in rats during the postnatal period was studied in vitro and in vivo after exposure of rat pups to antenatal acute hypobaric hypoxia at the stage of organogenesis (day 9–10 of gestation). Cultured cardiomyocytes from rat pups exposed to antenatal hypoxia were characterized by increased rate of contractions and decreased reactivity to norepinephrine. Heart rate elevation, predominance of sympathetic influences on cardiac activity, and significant increase in norepinephrine concentration in the cerebral cortex were found in freely moving animals exposed to antenatal hypoxia. Our results indicate that hypoxia at the stage of organogenesis modulated cardiac activity during the postnatal period, which manifested at the level of effector structures in the heart and activity of regulatory systems.

Thiamine Induces Long-Term Changes in Amino Acid Profiles and Activities of 2-Oxoglutarate and 2-Oxoadipate Dehydrogenases in Rat Brain.

Molecular mechanisms of long-term changes in brain metabolism after thiamine administration (single i.p. injection, 400 mg/kg) were investigated. Protocols for discrimination of the activities of the thiamine diphosphate (ThDP)-dependent 2-oxoglutarate and 2-oxoadipate dehydrogenases were developed to characterize specific regulation of the multienzyme complexes of the 2-oxoglutarate (OGDHC) and 2-oxoadipate (OADHC) dehydrogenases by thiamine. The thiamine-induced changes depended on the brain-region-specific expression of the ThDP-dependent dehydrogenases. In the cerebral cortex, the original levels of OGDHC and OADHC were relatively high and not increased by thiamine, whereas in the cerebellum thiamine upregulated the OGDHC and OADHC activities, whose original levels were relatively low. The effects of thiamine on each of the complexes were different and associated with metabolic rearrangements, which included (i) the brain-region-specific alterations of glutamine synthase and/or glutamate dehydrogenase and NADP+-dependent malic enzyme, (ii) the brain-region-specific changes of the amino acid profiles, and (iii) decreased levels of a number of amino acids in blood plasma. Along with the assays of enzymatic activities and average levels of amino acids in the blood and brain, the thiamine-induced metabolic rearrangements were assessed by analysis of correlations between the levels of amino acids. The set and parameters of the correlations were tissue-specific, and their responses to the thiamine treatment provided additional information on metabolic changes, compared to that gained from the average levels of amino acids. Taken together, the data suggest that thiamine decreases catabolism of amino acids by means of a complex and long-term regulation of metabolic flux through the tricarboxylic acid cycle, which includes coupled changes in activities of the ThDP-dependent dehydrogenases of 2-oxoglutarate and 2-oxoadipate and adjacent enzymes.

Effects of single intranasal administration of obestatin fragments on the body weight and feeding and drinking behaviors.

336 Lately, there has been a growing body of evidence about endogenous peptide regulators of appetite. Par ticular attention among them is paid to obestatin, which is a derivative of preproghrelin and composed of 23 amino acid residues [1]. An anorexigenic effect of obestatin has been shown in a number of studies [1, 2]. However, the study of anorexigenic effects of obestatin fragments are of a special interest. According to some data, fragment 1–13 has the strongest anorexigenic effect [3]; according to others, fragment 11–23 has [4]. There has been almost no analysis of the effects of other fragments. Therefore, we have investigated the effects of different obestatin fragments on body weight changes and the intake of food and water given ad libi tum. For our study, we chose fragments 1–4, 5–10, and 10–15 as less studied ones and fragment 11–23 whose anorexigenic effect had been shown earlier.

Glucose deprivation potentiates toxicity of ouabain and glutamate in cortical neurons cultured for different time periods

The addition of glutamate (Glu) to culture medium for 24 hours induced the dose-dependent death of rat cortical neurons cultured for 9–10 days and did not affect neurons cultured for 4–5 days; this suggests that the later neurons are neurochemically immature. In mature cultures, glucose deprivation (GD) enhanced the toxic effect of low Glu concentrations by 15% and did not influence the toxicity of high concentrations of this neuromediator. In immature cultures, GD potentiated the Glu effect independent of the concentration of this neuromediator. Inhibition of Na+/K+ -ATPase induced the death of some of the neurons. In the presence of a normal level of glucose, ouabain decreased the viability of mature and immature neurons to 67 ± 4 % and 79 ± 5%, respectively, and its presence during GD diminished viability to 28 ± 4 % and 56 ± 3%, respectively. The toxicity of ouabain was substantially attenuated when ionotropic glutamate receptors were blocked by MK-801. GD alone caused no significant increase in the death of these cells, even after a 3-hour incubation. Thus, GD strongly increases the susceptibility of neurons to the toxicity mediated by the activation of the NMDA subtype of ionotropic Glu receptors, even in the case of neurochemically immature neurons.

Cellular thiamine status is coupled to function of mitochondrial 2-oxoglutarate dehydrogenase

Decreased thiamine and reduced activity of thiamine diphosphate (ThDP)-dependent 2-oxoglutarate dehydrogenase (OGDH) cause neurodegeneration. We hypothesized on concerted cell-specific regulation of the thiamine metabolism and ThDP-dependent reactions. We identified a smaller thiamine pool, a lower expression of the mitochondrial ThDP transporter, and a higher expression of OGDH in rat astrocytes versus neuroblastoma N2A. According to the data, the astrocytic OGDH may be up-regulated by an increase in intracellular ThDP, while the neuroblastomal OGDH functions at full ThDP saturation. Indeed, in rat astrocytes and brain cortex, OGDH inhibition by succinyl phosphonate (SP) enlarged the pool of thiamine compounds. Increased ThDP level in response to the OGDH inhibition presumably up-regulated the enzyme to compensate for a decrease in reducing power which occurred in SP-treated astrocytes. Under the same SP treatment of N2A cells, their thiamine pool and reducing power were unchanged, although SP action was evident from accumulation of glutamate. The presented data indicate that functional interplay between OGDH, other proteins of the tricarbocylic acid cycle and proteins of thiamine metabolism is an important determinant of physiology-specific networks and their homeostatic mechanisms.

Directed regulation of multienzyme complexes of 2-oxo acid dehydrogenases using phosphonate and phosphinate analogs of 2-oxo acids

Abstract2-Oxo acid dehydrogenase complexes are important metabolic checkpoints functioning at the intercept of sugar and amino acid degradation. This review presents a short summary of architectural, catalytic, and regulatory principles of the complexes structure and function, based on recent advances in studies of well-characterized family members. Special attention is given to use of synthetic phosphonate and phosphinate analogs of 2-oxo acids as selective and efficient inhibitors of the cognate complexes in biological systems of bacterial, plant, and animal origin. We summarize our own results concerning the application of synthetic analogs of 2-oxo acids in situ and in vivo to reveal functional interactions between 2-oxo acid dehydrogenase complexes and other components of metabolic networks specific to different cells and tissues. Based on our study of glutamate excitotoxicity in cultured neurons, we show how a modulation of metabolism by specific inhibition of its key reaction may be employed to correct pathologies. This approach is further developed in our study on the action of the phosphonate analog of 2-oxoglutarate in animals. The study revealed that upregulation of 2-oxoglutarate dehydrogenase complex is involved in animal stress response and may provide increased resistance to damaging effects, underlying so-called preconditioning. The presented analysis of published data suggests synthetic inhibitors of metabolic checkpoints as promising tools to solve modern challenges of systems biology, metabolic engineering, and medicine.

Changes in feeding and drinking motivations and glucose content in male rats after single or chronic administration of obestatin or its fragment (1–4)

Obestatin is an endogenous regulator of appetite with anorexigenic action. In this study, we analyzed the effects of single and chronic intranasal administra� tions of obestatin or its fragment (1–4) at a dose of 300 nmol/kg on the glucose content of blood and food and water motivation under the conditions of depriva�