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The risk of developing diabetes and cardiometabolic disorders is associated with increased levels of alpha-aminoadipic acid and disturbances in the metabolism of branched-chain amino acids. The side effects of the widely used antidiabetic drug metformin include impaired degradation of branched-chain amino acids and inhibition of intracellular thiamin transport. These effects may be interconnected, as thiamine deficiency impairs the functioning of thiamine diphosphate (ThDP)-dependent dehydrogenases of 2-oxo acids involved in amino acids degradation, while diabetes is often associated with perturbed thiamine status. In this work, we investigate the action of metformin in rats with impaired thiamine availability. The reduction in the thiamine influx is induced by simultaneous administration of the thiamine transporters inhibitors metformin and amprolium. After 24 days of combined metformin/amprolium administration, no significant changes in the total brain levels of ThDP or activities of ThDP-dependent enzymes of central metabolism are observed, but the affinities of transketolase and 2-oxoglutarate dehydrogenase to ThDP increase. The treatment also significantly elevates the brain levels of free amino acids and ammonia, reduces the antioxidant defense, and alters the sympathetic/parasympathetic regulation, which is evident from changes in the ECG and behavioral parameters. Strong positive correlations between brain ThDP levels and contents of ammonia, glutathione disulfide, alpha-aminoadipate, glycine, citrulline, and ethanolamine are observed in the metformin/amprolium-treated rats, but not in the control animals. Analysis of the obtained data points to a switch in the metabolic impact of ThDP from the antioxidant and nitrogen-sparing in the control rats to the pro-oxidant and hyperammonemic in the metformin/amprolium-treated rats. As a result, metformin administration along with the amprolium-reduced thiamine supply significantly perturb the metabolism of amino acids in the rat brain, altering behavioral and ECG parameters.

Charcot-Marie-Tooth (CMT) neuropathy is a polygenic disorder of peripheral nerves with no effective cure. Thiamine (vitamin B1) is a neurotropic compound that improves neuropathies. Our pilot study characterizes therapeutic potential of daily oral administration of thiamine (100 mg) in CMT neuropathy and its molecular mechanisms. The patient hand grip strength was determined before and after thiamine administration along with the blood levels of the thiamine coenzyme form (thiamine diphosphate, ThDP), activities of endogenous holo-transketolase (without ThDP in the assay medium) and total transketolase (with ThDP in the assay medium), and transketolase activation by ThDP [1 - (holo-transketolase/total transketolase),%], corresponding to the fraction of ThDP-free apo-transketolase. Single cases of administration of sulbutiamine (200 mg) or benfotiamine (150 mg) reveal their effects on the assayed parameters within those of thiamine. Administration of thiamine or its pharmacological forms increased the hand grip strength in the CMT patients. Comparison of the thiamin status in patients with different forms of CMT disease to that of control subjects without diagnosed pathologies revealed no significant differences in the average levels of ThDP, holo-transketolase, or relative content of holo and apo forms of transketolase. However, the regulation of transketolase by thiamine/ThDP differed in the control and CMT groups: in the assay, ThDP activated transketolase from the control individuals, but not from CMT patients. Thiamine administration paradoxically decreased endogenous holo-transketolase in CMT patients; this effect was not observed in the control group. Correlation analysis revealed sex-specific differences in the relationship between the parameters of thiamine status in both the control subjects and patients with the CMT disease. Thus, our findings link physiological benefits of thiamine administration in CMT patients to changes in their thiamine status, in particular, the blood levels of ThDP and transketolase regulation.

Epilepsy is characterized by recurrent seizures due to a perturbed balance between glutamate and GABA neurotransmission. Our goal is to reveal the molecular mechanisms of the changes upon repeated challenges of this balance, suggesting knowledge-based neuroprotection. To address this goal, a set of metabolic indicators in the post-seizure rat brain cortex is compared before and after pharmacological kindling with pentylenetetrazole (PTZ). Vitamins B1 and B6 supporting energy and neurotransmitter metabolism are studied as neuroprotectors. PTZ kindling increases the seizure severity (1.3 fold, p < 0.01), elevating post-seizure rearings (1.5 fold, p = 0.03) and steps out of the walls (2 fold, p = 0.01). In the kindled vs. non-kindled rats, the post-seizure p53 level is increased 1.3 fold ( p = 0.03), reciprocating a 1.4-fold ( p = 0.02) decrease in the activity of 2-oxoglutarate dehydrogenase complex (OGDHC) controlling the glutamate degradation. Further, decreased expression of deacylases SIRT3 (1.4 fold, p = 0.01) and SIRT5 (1.5 fold, p = 0.01) reciprocates increased acetylation of 15 kDa proteins 1.5 fold ( p < 0.01). Finally, the kindling abrogates the stress response to multiple saline injections in the control animals, manifested in the increased activities of the pyruvate dehydrogenase complex, malic enzyme, glutamine synthetase and decreased malate dehydrogenase activity. Post-seizure animals demonstrate correlations of p53 expression to the levels of glutamate (r = 0.79, p = 0.05). The correlations of the seizure severity and duration to the levels of GABA (r = 0.59, p = 0.05) and glutamate dehydrogenase activity (r = 0.58, p = 0.02), respectively, are substituted by the correlation of the seizure latency with the OGDHC activity (r = 0.69, p < 0.01) after the vitamins administration, testifying to the vitamins-dependent impact of the kindling on glutamate/GABA metabolism. The vitamins also abrogate the correlations of behavioral parameters with seizure duration (r 0.53-0.59, p < 0.03). Thus, increased seizures and modified post-seizure behavior in rats after PTZ kindling are associated with multiple changes in the vitamin-dependent brain metabolism of amino acids, linked to key metabolic regulators: p53, OGDHC, SIRT3 and SIRT5.

Mitochondrial pyruvate dehydrogenase complex (PDHC) is essential for brain glucose and neurotransmitter metabolism, which is dysregulated in many pathologies. Using specific inhibitors of PDHC in vivo, we determine biochemical and physiological responses to PDHC dysfunction. Dose dependence of the responses to membrane-permeable dimethyl acetylphosphonate (AcPMe 2 ) is non-monotonous. Primary decreases in glutathione and its redox potential, methionine, and ethanolamine are alleviated with increasing PDHC inhibition, the alleviation accompanied by physiological changes. A comparison of 39 brain biochemical parameters after administration of four phosphinate and phosphonate analogs of pyruvate at a fixed dose of 0.1 mmol/kg reveals no primary, but secondary changes, such as activation of 2-oxoglutarate dehydrogenase complex (OGDHC) and decreased levels of glutamate, isoleucine and leucine. The accompanying decreases in freezing time are most pronounced after administration of methyl acetylphosphinate and dimethyl acetylphosphonate. The PDHC inhibitors do not significantly change the levels of PDHA1 expression and phosphorylation, sirtuin 3 and total protein acetylation, but increase total protein succinylation and glutarylation, affecting sirtuin 5 expression. Thus, decreased production of the tricarboxylic acid cycle substrate acetyl-CoA by inhibited PDHC is compensated by increased degradation of amino acids through the activated OGDHC, increasing total protein succinylation/glutarylation. Simultaneously, parasympathetic activity and anxiety indicators decrease.

Abnormal energy expenditure during seizures and metabolic regulation through post-translational protein acylation suggest acylation as a therapeutic target in epilepsy. Our goal is to characterize an interplay between the brain acylation system components and their changes after seizures. In a rat model of pentylenetetrazole (PTZ)-induced epilepsy, we quantify 43 acylations in 29 cerebral cortex proteins; levels of NAD + ; expression of NAD + -dependent deacylases (SIRT2, SIRT3, SIRT5); activities of the acyl-CoA-producing/NAD + -utilizing complexes of 2-oxoacid dehydrogenases. Compared to the control group, acylations of 14 sites in 11 proteins are found to differ significantly after seizures, with six of the proteins involved in glycolysis and energy metabolism. Comparing the single and chronic seizures does not reveal significant differences in the acylations, pyruvate dehydrogenase activity, SIRT2 expression or NAD + . On the contrary, expression of SIRT3, SIRT5 and activity of 2-oxoglutarate dehydrogenase (OGDH) decrease in chronic seizures vs. a single seizure. Negative correlations between the protein succinylation/glutarylation and SIRT5 expression, and positive correlations between the protein acetylation and SIRT2 expression are shown. Our findings unravel involvement of SIRT5 and OGDH in metabolic adaptation to seizures through protein acylation, consistent with the known neuroprotective role of SIRT5 and contribution of OGDH to the Glu/GABA balance perturbed in epilepsy.

In vitro and in cell cultures, succinyl phosphonate (SP) and adipoyl phosphonate (AP) selectively target dehydrogenases of 2-oxoglutarate (OGDH, encoded by OGDH/OGDHL ) and 2-oxoadipate (OADH, encoded by DHTKD1 ), respectively. To assess the selectivity in animals, the effects of SP, AP, and their membrane-penetrating triethyl esters (TESP and TEAP) on the rat brain metabolism and animal physiology are compared. Opposite effects of the OGDH and OADH inhibitors on activities of OGDH, malate dehydrogenase, glutamine synthetase, and levels of glutamate, lysine, citrulline, and carnosine are shown to result in distinct physiological responses. ECG is changed by AP/TEAP, whereas anxiety is increased by SP/TESP. The potential role of the ester moiety in the uncharged precursors of the 2-oxo acid dehydrogenase inhibitors is estimated. TMAP is shown to be less efficient than TEAP, in agreement with lower lipophilicity of TMAP vs . TEAP. Non-monotonous metabolic and physiological impacts of increasing OADH inhibition are revealed. Compared to the non-treated animals, strong inhibition of OADH decreases levels of tryptophan and beta-aminoisobutyrate and activities of malate dehydrogenase and pyruvate dehydrogenase, increasing the R-R interval of ECG. Thus, both metabolic and physiological actions of the OADH-directed inhibitors AP/TEAP are different from those of the OGDH-directed inhibitors SP/TESP, with the ethyl ester being more efficient than methyl ester., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bunik, Artiukhov, Kazantsev, Aleshin, Boyko, Ksenofontov, Lukashev and Graf.)

Background: The DHTKD1 -encoded 2-oxoadipate dehydrogenase (OADH) oxidizes 2-oxoadipate-a common intermediate of the lysine and tryptophan catabolism. The mostly low and cell-specific flux through these pathways, and similar activities of OADH and ubiquitously expressed 2-oxoglutarate dehydrogenase (OGDH), agree with often asymptomatic phenotypes of heterozygous mutations in the DHTKD1 gene. Nevertheless, OADH/ DHTKD1 are linked to impaired insulin sensitivity, cardiovascular disease risks, and Charcot-Marie-Tooth neuropathy. We hypothesize that systemic significance of OADH relies on its generation of glutaryl residues for protein glutarylation. Using pharmacological inhibition of OADH and the animal model of spinal cord injury (SCI), we explore this hypothesis., Methods: The weight-drop model of SCI, a single intranasal administration of an OADH-directed inhibitor trimethyl adipoyl phosphonate (TMAP), and quantification of the associated metabolic changes in the rat brain employ established methods., Results: The TMAP-induced metabolic changes in the brain of the control, laminectomized (LE) and SCI rats are long-term and (patho)physiology-dependent. Increased glutarylation of the brain proteins, proportional to OADH expression in the control and LE rats, represents a long-term consequence of the OADH inhibition. The proportionality suggests autoglutarylation of OADH, supported by our mass-spectrometric identification of glutarylated K155 and K818 in recombinant human OADH. In SCI rats, TMAP increases glutarylation of the brain proteins more than OADH expression, inducing a strong perturbation in the brain glutathione metabolism. The redox metabolism is not perturbed by TMAP in LE animals, where the inhibition of OADH increases expression of deglutarylase sirtuin 5. The results reveal the glutarylation-imposed control of the brain glutathione metabolism. Glutarylation of the ODP2 subunit of pyruvate dehydrogenase complex at K451 is detected in the rat brain, linking the OADH function to the brain glucose oxidation essential for the redox state. Short-term inhibition of OADH by TMAP administration manifests in increased levels of tryptophan and decreased levels of sirtuins 5 and 3 in the brain., Conclusion: Pharmacological inhibition of OADH affects acylation system of the brain, causing long-term, (patho)physiology-dependent changes in the expression of OADH and sirtuin 5, protein glutarylation and glutathione metabolism. The identified glutarylation of ODP2 subunit of pyruvate dehydrogenase complex provides a molecular mechanism of the OADH association with diabetes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Boyko, Karlina, Zavileyskiy, Aleshin, Artiukhov, Kaehne, Ksenofontov, Ryabov, Graf, Tramonti and Bunik.)

Oxidized nicotinamide adenine dinucleotide (NAD + ) is a biological molecule of systemic importance. Essential role of NAD + in cellular metabolism relies on the substrate action in various redox reactions and cellular signaling. This work introduces an efficient enzymatic assay of NAD + content in human blood using recombinant formate dehydrogenase (FDH, EC 1.2.1.2), and demonstrates its diagnostic potential, comparing NAD + content in the whole blood of control subjects and patients with cardiac or neurological pathologies. In the control group ( n = 22, 25-70 years old), our quantification of the blood concentration of NAD + (18 μM, minimum 15, max 23) corresponds well to NAD + quantifications reported in literature. In patients with demyelinating neurological diseases ( n = 10, 18-55 years old), the NAD + levels significantly ( p < 0.0001) decrease (to 14 μM, min 13, max 16), compared to the control group. In cardiac patients with the heart failure of stage II and III according to the New York Heart Association (NYHA) functional classification ( n = 24, 42-83 years old), the blood levels of NAD + (13 μM, min 9, max 18) are lower than those in the control subjects ( p < 0.0001) or neurological patients ( p = 0.1). A better discrimination of the cardiac and neurological patients is achieved when the ratios of NAD + to the blood creatinine levels, mean corpuscular volume or potassium ions are compared. The proposed NAD + assay provides an easy and robust tool for clinical analyses of an important metabolic indicator in the human blood., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Balashova, Zavileyskiy, Artiukhov, Shaposhnikov, Sidorova, Tishkov, Tramonti, Pometun and Bunik.)

2-Oxoacids are involved in a number of important metabolic processes and can be used as biomarkers in some human diseases. A new optimized method for quantification of 2,4-dinitrophenylhydrazine derivatives of 2-oxoacids using high-performance liquid chromatography was developed based on available techniques for quantification of 2-oxoacids in mammalian brain. The use of the 2,4-dinitrophenylhydrazine derivatives of 2-oxoacids was shown to be more advantageous in comparison with the previously used phenylhydrazine derivatives, due to a high chemical stability of the former. Here, we determined the concentrations of pyruvate, glyoxylate, 2-oxoglutarate, 2-oxomalonate, and 4-methylthio-2-oxobutyrate in the methanol/acetic acid extracts of the rat brain using the developed method, as well discussed the procedures for the sample preparation in analysis of mammalian brain extracts. The validation parameters of the method demonstrated that the quantification limits for each of the analyzed of 2-oxoacids was 2 nmol/mg tissue. The developed method facilitates identification of subtle changes in the tissue and cellular content of 2-oxoacids as (patho)physiological biomarkers of metabolism in mammalian tissues.

Hypoxia is damaging to the fetus, but the developmental impact may vary, with underlying molecular mechanisms unclear. We demonstrate the dependence of physiological and biochemical effects of acute prenatal hypoxia (APH) on sex and gestational age. Compared to control rats, APH on the 10th day of pregnancy (APH-10) increases locomotion in both the male and female offspring, additionally increasing exploratory activity and decreasing anxiety in the males. Compared to APH-10, APH on the 20th day of pregnancy (APH-20) induces less behavioral perturbations. ECG is changed similarly in all offspring only by APH-10. Sexual dimorphism in the APH outcome on behavior is also observed in the brain acetylation system and 2-oxoglutarate dehydrogenase reaction, essential for neurotransmitter metabolism. In view of the perturbed behavior, more biochemical parameters in the brains are assessed after APH-20. Of the six enzymes, APH-20 significantly decreases the malic enzyme activity in both sexes. Among 24 amino acids and dipeptides, APH-20 increases the levels of only three amino acids (Phe, Thr, and Trp) in male offspring, and of seven amino acids (Glu, Gly, Phe, Trp, Ser, Thr, Asn) and carnosine in the female offspring. Thus, a higher reactivity of the brain metabolism to APH stabilizes the behavior. The behavior and brain biochemistry demonstrate sexually dimorphic responses to APH at both gestational stages, whereas the APH effects on ECG depend on gestational age rather than sex.

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.

The glutarylation of lysine residues in proteins attracts attention as a possible mechanism of metabolic regulation, perturbed in pathologies. The visualization of protein glutarylation by antibodies specific to ε-glutaryl-lysine residues may be particularly useful to reveal pathogenic mutations in the relevant enzymes. We purified such antibodies from the rabbit antiserum, obtained after sequential immunization with two artificially glutarylated proteins, using affinity chromatography on ε-glutaryl-lysine-containing sorbents. Employing these anti(ε-glutaryl-lysine)-antibodies for the immunoblotting analysis of rat tissues and mitochondria has demonstrated the sample-specific patterns of protein glutarylation. The study of the protein glutarylation in rat tissue homogenates revealed a time-dependent fragmentation of glutarylated proteins in these preparations. The process may complicate the investigation of potential changes in the acylation level of specific protein bands when studying time-dependent effects of the acylation regulators. In the rat brain, the protein glutarylation, succinylation and acetylation patterns obtained upon the immunoblotting of the same sample with the corresponding antibodies are shown to differ. Specific combinations of molecular masses of major protein bands in the different acylation patterns confirm the selectivity of the anti(ε-glutaryl-lysine)-antibodies obtained in this work. Hence, our affinity-purified anti(ε-glutaryllysine)-antibodies provide an effective tool to characterize protein glutarylation, revealing its specific pattern, compared to acetylation and succinylation, in complex protein mixtures.

The disturbed metabolism of vitamins B1 or B6, which are essential for neurotransmitters homeostasis, may cause seizures. Our study aims at revealing therapeutic potential of vitamins B1 and B6 by estimating the short- and long-term effects of their combined administration with the seizure inductor pentylenetetrazole (PTZ). The PTZ dose dependence of a seizure and its parameters according to modified Racine's scale, along with delayed physiological and biochemical consequences the next day after the seizure are assessed regarding sexual dimorphism in epilepsy. PTZ sensitivity is stronger in the female than the male rats. The next day after a seizure, sex differences in behavior and brain biochemistry arise. The induced sex differences in anxiety and locomotor activity correspond to the disappearance of sex differences in the brain aspartate and alanine, with appearance of those in glutamate and glutamine. PTZ decreases the brain malate dehydrogenase activity and urea in the males and the phenylalanine in the females. The administration of vitamins B1 and B6 24 h before PTZ delays a seizure in female rats only. This desensitization is not observed at short intervals (0.5-2 h) between the administration of the vitamins and PTZ. With the increasing interval, the pyridoxal kinase (PLK) activity in the female brain decreases, suggesting that the PLK downregulation by vitamins contributes to the desensitization. The delayed effects of vitamins and/or PTZ are mostly sex-specific and interacting. Our findings on the sex differences in sensitivity to epileptogenic factors, action of vitamins B1/B6 and associated biochemical events have medical implications.

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.

Phosphonate analogs of pyruvate and 2-oxoglutarate are established specific inhibitors of cognate 2-oxo acid dehydrogenases. The present work develops application of this class of compounds to specific in vivo inhibition of 2-oxoglutarate dehydrogenase (OGDH) and its isoenzyme, 2-oxoadipate dehydrogenase (OADH). The isoenzymes-enriched preparations from the rat tissues with different expression of OADH and OGDH are used to characterize their interaction with 2-oxoglutarate (OG), 2-oxoadipate (OA) and the phosphonate analogs. Despite a 100-fold difference in the isoenzymes ratio in the heart and liver, similar Michaelis saturations by OG are inherent in the enzyme preparations from these tissues ( K m O G = 0.45 ± 0.06 and 0.27 ± 0.026 mM, respectively), indicating no significant contribution of OADH to the OGDH reaction, or similar affinities of the isoenzymes to OG. However, the preparations differ in the catalysis of OADH reaction. The heart preparation, where OADH/OGDH ratio is ≈ 0.01, possesses low-affinity sites to OA ( K m O A = 0.55 ± 0.07 mM). The liver preparation, where OADH/OGDH ratio is ≈ 1.6, demonstrates a biphasic saturation with OA: the low-affinity sites ( K m , 2 O A = 0.45 ± 0.12 mM) are similar to those of the heart preparation; the high-affinity sites ( K m , 1 O A = 0.008 ± 0.001 mM), revealed in the liver preparation only, are attributed to OADH. Phosphonate analogs of C5-C7 dicarboxylic 2-oxo acids inhibit OGDH and OADH competitively to 2-oxo substrates in all sites. The high-affinity sites for OA are affected the least by the C5 analog (succinyl phosphonate) and the most by the C7 one (adipoyl phosphonate). The opposite reactivity is inherent in both the low-affinity OA-binding sites and OG-binding sites. The C6 analog (glutaryl phosphonate) does not exhibit a significant preference to either OADH or OGDH. Structural analysis of the phosphonates binding to OADH and OGDH reveals the substitution of a tyrosine residue in OGDH for a serine residue in OADH among structural determinants of the preferential binding of the bulkier ligands to OADH. The consistent kinetic and structural results expose adipoyl phosphonate as a valuable pharmacological tool for specific in vivo inhibition of the DHTKD1 -encoded OADH, a new member of mammalian family of 2-oxo acid dehydrogenases, up-regulated in some cancers and associated with diabetes and obesity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Artiukhov, Kazantsev, Lukashev, Bellinzoni and Bunik.)

The medical significance of NAD + -dependent metabolic regulation acquires increasing attention, demanding rapid and clinically feasible quantification of NAD + in complex biological samples. Here we describe the usage of formate dehydrogenase for a straightforward and highly specific fluorometric assay of NAD + in tissue extracts, not requiring chromatographic separation of nucleotides. The assay employs the irreversible reaction of formate oxidation coupled to NAD + reduction, catalyzed by the enzyme which has high affinity and specificity to NAD + , and is stable under a variety of conditions. The assay reliably quantifies NAD + in the methanol extracts of the rat brain cortex and mitochondria., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Unlike the OGDH-encoded 2-oxoglutarate dehydrogenase (OGDH), which is an essential enzyme present in all animal tissues, expression of the DHTKD1-encoded isoenzyme, 2-oxoadipate dehydrogenase (OADH), depends on a number of factors, and mutant DHTKD1 phenotypes are rarely manifested. Physiological significance of OADH is also obscured by the fact that both isoenzymes transform 2-oxoglutarate and 2-oxoadipate. By analogy with other members of the 2-oxo acid dehydrogenases family, OADH is assumed to be a component of the multienzyme complex that catalyzes oxidative decarboxylation of 2-oxoadipate. This study aims at molecular characterization of OADH from animal tissues. Phylogenetic analysis of 2-oxo acid dehydrogenases reveals OADH only in animals and Dictyostelium discoideum slime mold, within a common branch with bacterial OGDH. Examination of partially purified animal OADH by immunoblotting and mass spectrometry identifies two OADH isoforms with molecular weights of about 130 and 70 kDa. These isoforms are not observed upon the expression of human DHTKD1 protein in either bacterial or yeast system, where the synthesized OADH is of expected molecular weight (about 100 kDa). Thus, the OADH isoforms present in animal tissues, may result from the animal-specific regulation of the DHTKD1 expression and/or posttranslational modifications of the encoded protein. Mapping of the peptides identified in the OADH preparations, onto the protein structure suggests that the 70-kDa isoform is truncated at the N-terminus, but retains the active site. Since the N-terminal domain of OGDH is required for the formation of the multienzyme complex, it is possible that the 70-kDa isoform catalyzes non-oxidative transformation of dicarboxylic 2-oxo acids that does not require the multienzyme structure. In this case, the ratio of the OADH isoforms in animal tissues may correspond to the ratio between the oxidative and non-oxidative decarboxylation of 2-oxoadipate.

The biological significance of the DHTKD1-encoded 2-oxoadipate dehydrogenase (OADH) remains obscure due to its catalytic redundancy with the ubiquitous OGDH-encoded 2-oxoglutarate dehydrogenase (OGDH). In this work, metabolic contributions of OADH and OGDH are discriminated by exposure of cells/tissues with different DHTKD1 expression to the synthesized phosphonate analogues of homologous 2-oxodicarboxylates. The saccharopine pathway intermediates and phosphorylated sugars are abundant when cellular expressions of DHTKD1 and OGDH are comparable, while nicotinate and non-phosphorylated sugars are when DHTKD1 expression is order(s) of magnitude lower than that of OGDH. Using succinyl, glutaryl and adipoyl phosphonates on the enzyme preparations from tissues with varied DHTKD1 expression reveals the contributions of OADH and OGDH to oxidation of 2-oxoadipate and 2-oxoglutarate in vitro. In the phosphonates-treated cells with the high and low DHTKD1 expression, adipate or glutarate, correspondingly, are the most affected metabolites. The marker of fatty acid β-oxidation, adipate, is mostly decreased by the shorter, OGDH-preferring, phosphonate, in agreement with the known OGDH dependence of β-oxidation. The longest, OADH-preferring, phosphonate mostly affects the glutarate level. Coupled decreases in sugars and nicotinate upon the OADH inhibition link the perturbation in glucose homeostasis, known in OADH mutants, to the nicotinate-dependent NAD metabolism.

To study the mechanisms of the non-coenzyme action of thiamine and its diphosphate (ThDP) on brain proteins, proteins of acetone extract of bovine brain synaptosomes or the homogenate of rat brain cortex were subjected to affinity chromatography on thiamine-modified Sepharose. In the step-wise eluates by thiamine (at pH 7.4 or 5.6), NaCl, and urea, the occurrence of glutamate dehydrogenase (GDH) and isoenzymes of malate dehydrogenase (MDH) along with the influence of thiamine and/or ThDP on the enzymatic activities were characterized using mass spectrometry and kinetic experiments. Maximal activation of the malate dehydrogenase reaction by thiamine is observed after the protein elution with the acidic thiamine solution, which does not elute the MDH1 isoenzyme. Effects of exogenous thiamine or ThDP on the GDH activity may depend on endogenous enzyme regulators. For example, thiamine and/or ThDP activate the brain GDH in eluates from thiamine-Sepharose but inhibit the enzyme in the crude preparations applied to the sorbent. Inhibition of GDH by ThDP is observed using the ADP-activated enzyme. Compared to the affinity chromatography employing the elution by thiamine at pH 7.4, the procedure at pH 5.6 decreases the activation of GDH by thiamine (but not ThDP) in the eluates with NaCl and urea. Simultaneously, the MDH2 content and total GDH activity are higher after the affinity elution at pH 5.6 than at pH 7.4, suggesting the role of the known interaction of GDH with MDH2 in stabilizing the activity of GDH and in the regulation of GDH by thiamine. The biological potential of thiamine-dependent regulation of the brain GDH is confirmed in vivo by demonstration of changes in regulatory properties of GDH after administration of a high dose of thiamine to rats. Bioinformatics analysis of the thiamine-eluted brain proteins shows a specific enrichment of their annotation terms with "phosphoprotein", "acetylation", and "methylation". The relationship between thiamine and the posttranslational modifications in brain may contribute to the neuroprotective effects of high doses of thiamine, including the regulation of oxidation of the major excitatory neurotransmitter in brain - glutamate.

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.

2-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.

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.

The mathematical modeling and clinical testing of results of usage of low molar segments as supporting elements after tooth preserving surgery have been made. After hemisection and crown-radicular separation the critical load for low molar segments is more than 30 kg; after root amputation the critical load for low molar segments is more than 18 kg. The mathematical modeling, clinical observation, and X-ray photography have shown that the remained supporting system of low molars provides a full functioning of the teeth-jaw system after crown radicular separation and hemisection tooth preserving surgery.