L. K. Trofimova

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.

Consequences of the α-ketoglutarate dehydrogenase inhibition for neuronal metabolism and survival: implications for neurodegenerative diseases.

Neurodegenerative diseases are accompanied by reduced activity of mitochondrial α-ketoglutarate dehydrogenase multienzyme complex (KGDHC). We present a new cellular model to study molecular mechanisms of this association. By application of the highly specific and efficient inhibitor of KGDHC, succinyl phosphonate (SP), to cultured neurons, we characterized the concentration- and time-dependent consequences of decreased KGDHC activity for neuronal metabolism and viability. Metabolic profiling of SP-treated neurons established accumulation of α-ketoglutarate and pyruvate as indicators of the KGDHC inhibition and ensuing impairment of pyruvate oxidation in the tricarboxylic acid cycle. Concomitant increases in alanine, glutamate and γ-aminobutyrate indicated a scavenging of the accumulated pyruvate and α-ketoglutarate by transamination and further decarboxylation of glutamate. Changes among other amino acids were in accordance with their potential to react with α-ketoglutarate or products of its transamination and serve as fuel compensating for the KGDHC block. Disturbances in neuronal amino acid pool were accompanied by changed polyamines, decreased total protein and increased thymine, suggesting increased catabolism of amino acids to decrease translation and affect DNA turnover/repair. The ensuing ATP salvage was observed as the paradoxical increase in neuronal ATP by mitochondrial inhibitor SP. Extensive exposure of neurons to SP reduced viability, as revealed by both the ATP- and NAD(P)H-dependent viability tests. Thus, we provide experimental evidence on the KGDHC impairment as a cause of neurodegeneration and decipher underlying molecular mechanisms, exposing the key regulatory complex of the tricarboxylic acid cycle as a promising target for directed regulation of neuronal function and survival.

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.

Effects of GSM-Frequency Electromagnetic Radiation on Some Physiological and Biochemical Parameters in Rats

Single exposure of white outbred rats to electromagnetic radiation with a frequency 905 MHz (GSM frequency) for 2 h increased anxiety, reduced locomotor, orientation, and exploration activities in females and orientation and exploration activities in males. Glucocorticoid levels and antioxidant system activity increased in both males and females. In addition to acute effects, delayed effects of radiation were observed in both males and females 1 day after the exposure. These results demonstrated signifi cant effect of GSM-range radiation on the behavior and activity of stress-realizing and stress-limiting systems of the body.

Effect of antenatal hypoxia on age-specific dynamics of ECG parameters and content of biogenic amines in the central nervous system.

We studied the effect of acute antenatal hypoxia during the stages of progestation and early organogenesis on some ECG parameters and level of biogenic amines in brain structures in rats. The effect of acute hypoxic exposure during the organogenesis period on the studied parameters was more pronounced than the effect of acute hypoxic exposure during the progestation period. The shift of the autonomic balance towards the sympathetic regulation of cardiac activity is linked with increased content of biogenic amines in the brain stem and cortical structures.

Effect of Anorexigenic Peptide Obestatin on Platelet Aggregation and Osmotic Resistance of Erythrocytes

We studied the effects of the anorexigenic peptide obestatin on the coagulation system and blood rheology (by the parameters of platelet aggregation and osmotic resistance of erythrocytes) in vitro and in vivo. Obestatin inhibited in vitro platelet aggregation in the entire dose range and reduced osmotic resistance of erythrocytes in all doses except 300 nmol/kg (obestatin in a dose of 300 nmol/kg had no effect on this parameter). Similar to the results of in vitro experiments, intranasal, intraperitoneal, and subcutaneous administration of obestatin in a dose of 300 nmol/kg inhibited platelet aggregation and had no effect on the osmotic resistance of erythrocytes.

Transgeneration Effects of Antenatal Acute Hypoxia during Early Organogenesis

We studied the effect of acute hypobaric hypoxia in early organogenesis on physiological and behavioral parameters of second-generation albino rats. Antenatal acute hypoxia was followed by physical and sexual retardation, increase in the mortality rate, and behavioral changes in second-generation animals (hypoactivity of males and females on day 22 of life and hyperactivity of males on day 57 of life). Second-generation animals exhibited no gender differences in body weight and horizontal and vertical locomotor activity.

Influence of intermittent normobaric hypoxia during early organogenesis on the development of white rats

The influence of antenatal intermittent normobaric hypoxia during early organogenesis (days 9–10 of intrauterine development) on the physical development, vegetative balance, and antioxidant defense system of 60-day-old rats was studied. Antenatal exposure to intermittent hypoxia resulted in the impaired physical development of all offspring during the early 15-day postnatal period and caused changes in the vegetative balance of heart regulation, which were differently directed in males and females. Moreover, females that survived antenatal hypoxia had a decreased superoxide dismutase activity in the brain, compared to that in the control rats.

Positive correlation between rat brain glutamate concentrations and mitochondrial 2-oxoglutarate dehydrogenase activity

Glutamate is a key metabolite and major excitatory neurotransmitter, degraded through transformation to 2-oxoglutarate which is further catabolized by 2-oxoglutarate dehydrogenase complex (OGDHC). Both the glutamate excitotoxicity and impaired OGDHC activity are hallmarks of neurodegeneration. This work quantifies a relationship between the brain OGDHC activity and glutamate levels, assessing its diagnostic value to characterize (patho)physiology. A moderate to strong positive correlation of the two parameters determined under varied physiological settings (brain regions, seasons, gender, pregnancy, rat line), is revealed. Mitochondrial impairment (OGDHC inhibition or acute hypobaric hypoxia) decreases the interdependence, even when the parameter means do not change significantly. Compared to the cortex, the cerebellum exhibits a lower inter-individual glutamate variation and a weaker glutamate-OGDHC interdependence. Specific metabolism of the brain regions is also characterized by a positive correlation between glutamate and γ-aminobutyric acid (GABA) concentrations in the cortex but not in the cerebellum. In contrast, a strong positive correlation between glutamate and glutamine is present in both the cortex and cerebellum. The differences in metabolic correlations are in line with transcriptomics data which suggest that glutamate distribution between competitive pathways contributes to the brain-region-specific features of the interdependences of glutamate and OGDHC or GABA.