Α-ketoglutarate Dehydrogenase Activity Research Articles

Pristanic acid provokes lipid, protein, and DNA oxidative damage and reduces the antioxidant defenses in cerebellum of young rats.

Zellweger syndrome (ZS) and some peroxisomal diseases are severe inherited disorders mainly characterized by neurological symptoms and cerebellum abnormalities, whose pathogenesis is poorly understood. Biochemically, these diseases are mainly characterized by accumulation of pristanic acid (Prist) and other fatty acids in the brain and other tissues. In this work, we evaluated the in vitro influence of Prist on redox homeostasis by measuring lipid, protein, and DNA damage, as well as the antioxidant defenses and the activities of aconitase and α-ketoglutarate dehydrogenase in cerebellum of 30-day-old rats. The effect of Prist on DNA damage was also evaluated in blood of these animals. Some parameters were also evaluated in cerebellum from neonatal rats and in cerebellum neuronal cultures. Prist significantly increased malondialdehyde (MDA) levels and carbonyl formation and reduced sulfhydryl content and glutathione (GSH) concentrations in cerebellum of young rats. It also caused DNA strand damage in cerebellum and induced a high micronuclei frequency in blood. On the other hand, this fatty acid significantly reduced α-ketoglutarate dehydrogenase and aconitase activities in rat cerebellum. We also verified that Prist-induced increase of MDA levels was totally prevented by melatonin and attenuated by α-tocopherol but not by the nitric oxide synthase inhibitor N(ω)-nitro-L-arginine methyl ester, indicating the involvement of reactive oxygen species in this effect. Cerebellum from neonate rats also showed marked alterations of redox homeostasis, including an increase of MDA levels and a decrease of sulfhydryl content and GSH concentrations elicited by Prist. Finally, Prist provoked an increase of dichlorofluorescein (DCFH) oxidation in cerebellum-cultivated neurons. Our present data indicate that Prist compromises redox homeostasis in rat cerebellum and blood and inhibits critical enzymes of the citric acid cycle that are susceptible to free radical attack. The present findings may contribute to clarify the pathogenesis of the cerebellar alterations observed in patients affected by ZS and some peroxisomal disorders in which Prist is accumulated.

Acute intermittent porphyria causes hepatic mitochondrial energetic failure in a mouse model

Acute intermittent porphyria (AIP), an inherited hepatic disorder, is due to a defect of hydroxymethylbilane synthase (HMBS), an enzyme involved in heme biosynthesis. AIP is characterized by recurrent, life-threatening attacks at least partly due to the increased hepatic production of 5-aminolaevulinic acid (ALA). Both the mitochondrial enzyme, ALA synthase (ALAS) 1, involved in the first step of heme biosynthesis, which is closely linked to mitochondrial bioenergetic pathways, and the promise of an ALAS1 siRNA hepatic therapy in humans, led us to investigate hepatic energetic metabolism in Hmbs KO mice treated with phenobarbital. The mitochondrial respiratory chain (RC) and the tricarboxylic acid (TCA) cycle were explored in the Hmbs−/− mouse model. RC and TCA cycle were significantly affected in comparison to controls in mice treated with phenobarbital with decreased activities of RC complexes I (−52%, **p<0.01), II (−50%, **p<0.01) and III (−55%, *p<0.05), and decreased activity of α-ketoglutarate dehydrogenase (−64%, *p<0.05), citrate synthase (−48%, **p<0.01) and succinate dehydrogenase (−53%, *p<0.05). Complex II-driven succinate respiration was also significantly affected. Most of these metabolic alterations were at least partially restored after the phenobarbital arrest and heme arginate administration. These results suggest a cataplerosis of the TCA cycle induced by phenobarbital, caused by the massive withdrawal of succinyl-CoA by ALAS induction, such that the TCA cycle is unable to supply the reduced cofactors to the RC. This profound and reversible impact of AIP on mitochondrial energetic metabolism offers new insights into the beneficial effect of heme, glucose and ALAS1 siRNA treatments by limiting the cataplerosis of TCA cycle.

Burst of succinate dehydrogenase and α-ketoglutarate dehydrogenase activity in concert with the expression of genes coding for respiratory chain proteins underlies short-term beneficial physiological stress in mitochondria

Conditions for the realization in rats of moderate physiological stress (PHS) (30–120min) were selected, which preferentially increase adaptive restorative processes without adverse responses typical of harmful stress (HST). The succinate dehydrogenase (SDH) and α-ketoglutarate dehydrogenase (KDH) activity and the formation of reactive oxygen species (ROS) in mitochondria were measured in lymphocytes by the cytobiochemical method, which detects the regulation of mitochondria in the organism with high sensitivity.These mitochondrial markers undergo an initial 10–20-fold burst of activity followed by a decrease to a level exceeding the quiescent state 2–3-fold by 120min of PHS. By 30–60min, the rise in SDH activity was greater than in KDH activity, while the activity of KDH prevailed over that of SDH by 120min. The attenuation of SDH hyperactivity during PHS occurs by a mechanism other than oxaloacetate inhibition developed under HST.The dynamics of SDH and KDH activity corresponds to the known physiological replacement of adrenergic regulation by cholinergic during PHS, which is confirmed here by mitochondrial markers because their activity reflects these two types of nerve regulation, respectively. The domination of cholinergic regulation provides the overrestoration of expenditures for activity. In essence, this phenomenon corresponds to the training of the organism. It was first revealed in mitochondria after a single short-time stress episode.The burst of ROS formation was congruous with changes in SDH and KDH activity, as well as in ucp2 and cox3 expression, while the activity of SDH was inversely dependent on the expression of the gene of its catalytic subunit in the spleen. As the SDH activity enhanced, the expression of the succinate receptor decreased with subsequent dramatic rise when the activity was becoming lower.This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaption and therapy.

Production‐cycle‐related changes in indices of lysine catabolism in turkey liver

In typical turkey production diets, lysine is frequently the second limiting amino acid. Understanding its catabolism could provide opportunities to increase the efficiency of lysine use for protein synthesis. We hypothesize that indices of lysine catabolism in turkey liver vary throughout the production cycle. Two commercial strains of turkey, Hybrid and Nicolas, were analyzed 8 times over a period of 17 weeks (n=8 birds/sampling time/strain) for lysine α‐ketoglutarate (LKR) and saccharopine dehydrogenase (SDH) activity and mRNA abundance, in vitro lysine oxidation (LOX) and amino acid oxidase (AAOX) activity. We found differences in LKR mRNA (P&lt;0.01), SDH activity (P&lt;0.05) and LOX (P&lt;0.0001) and a trend for differences in LKR activity (P=0.09), SDH mRNA (P=0.07) and AAOX activity (P=0.06) throughout the production cycle. No consistent strain differences were detected for any variable. Interestingly, the average LKR and SDH activities across strains and weeks was 230 and 400 nmol per minute per gram of liver, respectively, as opposed to the average AAOX activity and LOX was 1.25 and 13.6 nmol per minute per gram liver, respectively. These data indicate that the saccharopine‐dependent pathway is the predominant pathway of lysine degradation in turkey liver and that indices of hepatic lysine catabolism vary throughout the production cycle. (Support; HATCH WVA 470, Virginia Poultry Growers Cooperative)

Acetyl-CoA deficit in brain mitochondria in experimental thiamine deficiency encephalopathy

Several pathologic conditions are known to cause thiamine deficiency, which induce energy shortages in all tissues, due to impairment of pyruvate decarboxylation. Brain is particularly susceptible to these conditions due to its high rate of glucose to pyruvate-driven energy metabolism. However, cellular compartmentalization of a key energy metabolite, acetyl-CoA, in this pathology remains unknown. Pyrithiamine-evoked thiamine deficiency caused no significant alteration in pyruvate dehydrogenase and 30% inhibition of α-ketoglutarate dehydrogenase activities in rat whole forebrain mitochondria. It also caused 50% reduction of the metabolic flux of pyruvate through pyruvate dehydrogenase, 78% inhibition of its flux through α-ketoglutarate dehydrogenase steps, and nearly 60% decrease of intramitochondrial acetyl-CoA content, irrespective of the metabolic state. State 3 caused a decrease in citrate and an increase in α-ketoglutarate accumulation. These alterations were more evident in thiamine-deficient mitochondria. Simultaneously thiamine deficiency caused no alteration of relative, state 3-induced increases in metabolic fluxes through pyruvate and α-ketoglutarate dehydrogenase steps. These data indicate that a shortage of acetyl-CoA in the mitochondrial compartment may be a primary signal inducing impairment of neuronal and glial cell functions and viability in the thiamine-deficient brain.

Changes in sympathetic and parasympathetic regulation connected with succinate dehydrogenase and α-ketoglutarate dehydrogenase activity in different physiological states of the organism

Changes in sympathetic and parasympathetic regulation connected with succinate dehydrogenase and α-ketoglutarate dehydrogenase activity in different physiological states of the organism

α-Ketoisocaproic acid and leucine provoke mitochondrial bioenergetic dysfunction in rat brain

Patients affected by maple syrup urine disease (MSUD) present severe neurological symptoms and brain abnormalities, whose pathoiphysiology is poorly known. In the present study we investigated the in vitro effects of leucine (Leu), α-ketoisocaproic acid (KIC) and α-hydroxyisovaleric acid (HIV), respectively, the branched-chain amino, keto and hydroxy acids that most accumulate in MSUD, on brain bioenergetic homeostasis, evaluating respiratory parameters obtained by oxygen consumption, membrane potential (∆Ψm), NAD(P)H content, swelling and citric acid cycle enzyme activities in mitochondrial preparations from rat forebrain using glutamate plus malate, succinate or α-ketoglutarate as respiratory substrates. KIC increased state 4 and decreased the respiratory control ratio with all substrates, in contrast with Leu and HIV. Furthermore, KIC and Leu, but not HIV, decreased state 3 using α-ketoglutarate. A KIC-induced selective inhibition of α-ketoglutarate dehydrogenase activity was also verified, with no alteration of the other citric acid cycle activities. The ADP/O ratio and the mitochondrial NAD(P)H levels were also reduced by KIC using glutamate/malate and α-ketoglutarate. In addition, KIC caused a reduction in the ∆Ψm when α-ketoglutarate was the substrate. Finally, KIC was not able to induce mitochondrial swelling. The present data indicate that KIC acts as an uncoupler of oxidative phosphorylation and as a metabolic inhibitor possibly through its inhibitory effect on α-ketoglutarate dehydrogenase activity, while Leu acts as a metabolic inhibitor. It is suggested that impairment of mitochondrial homeostasis caused by the major metabolites accumulating in MSUD may be involved in the neuropathology of this disease.

Rapid determination of tricarboxylic acid cycle enzyme activities in biological samples

BackgroundIn the last ten years, deficiencies in tricarboxylic acid cycle (TCAC) enzymes have been shown to cause a wide spectrum of human diseases, including malignancies and neurological and cardiac diseases. A prerequisite to the identification of disease-causing TCAC enzyme deficiencies is the availability of effective enzyme assays.ResultsWe developed three assays that measure the full set of TCAC enzymes. One assay relies on the sequential addition of reagents to measure succinyl-CoA ligase activity, followed by succinate dehydrogenase, fumarase and, finally, malate dehydrogenase. Another assay measures the activity of α-ketoglutarate dehydrogenase followed by aconitase and isocitrate dehydrogenase. The remaining assay measures citrate synthase activity using a standard procedure. We used these assays successfully on extracts of small numbers of human cells displaying various severe or partial TCAC deficiencies and on frozen heart homogenates from heterozygous mice harboring an SDHB gene deletion.ConclusionThis set of assays is rapid and simple to use and can immediately detect even partial defects, as the activity of each enzyme can be readily compared with one or more other activities measured in the same sample.

Effects of long-term oxygen treatment on α-ketoglutarate dehydrogenase activity and oxidative modifications in mitochondria of the guinea pig heart

ObjectiveOxygen therapy is used for the treatment of various diseases, but prolonged exposure to high concentrations of O2 is also associated with formation of free radicals and oxidative damage.MethodsIn the present study we compared α-ketoglutarate dehydrogenase (KGDH) activity and mitochondrial oxidative damage in the hearts of guinea pigs after long-term (17 and 60 h) oxygenation with 100% normobaric O2 and with partially negatively (O2 neg) or positively (O2 posit) ionized oxygen.ResultsInhalation of O2 led to significant loss in KGDH activity and thiol group content and accumulation of bityrosines. Inhalation of O2 neg was accompanied by more pronounced KGDH inhibition, possibly due to additional formation of protein-lipid conjugates. In contrast, O2 posit prevented loss in KGDH activity and diminished mitochondrial oxidative damage.ConclusionsThese findings suggest that oxygen treatment is associated with impairment of heart energy metabolism and support the view that inhalation of O2 posit optimizes the beneficial effects of oxygen therapy.

Rapamycin impairs metabolism-secretion coupling in rat pancreatic islets by suppressing carbohydrate metabolism

Rapamycin, an immunosuppressant used in human transplantation, impairs beta-cell function, but the mechanism is unclear. Chronic (24 h) exposure to rapamycin concentration dependently suppressed 16.7 mM glucose-induced insulin release from islets (1.65+/-0.06, 30 nM rapamycin versus 2.35+/-0.11 ng/islet per 30 min, control, n=30, P<0.01) without affecting insulin and DNA contents. Rapamycin also decreased alpha-ketoisocaproate-induced insulin release, suggesting reduced mitochondrial carbohydrate metabolism. ATP content in the presence of 16.7 mM glucose was significantly reduced in rapamycin-treated islets (13.42+/-0.47, rapamycin versus 16.04+/-0.46 pmol/islet, control, n=30, P<0.01). Glucose oxidation, which indicates the velocity of metabolism in the Krebs cycle, was decreased by rapamycin in the presence of 16.7 mM glucose (30.1+/-2.7, rapamycin versus 42.2+/-3.3 pmol/islet per 90 min, control, n=9, P<0.01). Immunoblotting revealed that the expression of complex I, III, IV, and V was not affected by rapamycin. Mitochondrial ATP production indicated that the respiratory chain downstream of complex II was not affected, but that carbohydrate metabolism in the Krebs cycle was reduced by rapamycin. Analysis of enzymes in the Krebs cycle revealed that activity of alpha-ketoglutarate dehydrogenase (KGDH), which catalyzes one of the slowest reactions in the Krebs cycle, was reduced by rapamycin (10.08+/-0.82, rapamycin versus 13.82+/-0.84 nmol/mg mitochondrial protein per min, control, n=5, P<0.01). Considered together, these findings indicate that rapamycin suppresses high glucose-induced insulin secretion from pancreatic islets by reducing mitochondrial ATP production through suppression of carbohydrate metabolism in the Krebs cycle, together with reduced KGDH activity.

Experimental evidence that ornithine and homocitrulline disrupt energy metabolism in brain of young rats

Tissue accumulation of ornithine (Orn), homocitrulline (Hcit), ammonia and orotic acid (Oro) is the biochemical hallmark of patients affected by hyperornithinemia–hyperammonemia–homocitrullinuria (HHH) syndrome, a disorder clinically characterized by neurological symptoms, whose pathophysiology is practically unknown. In the present study, we investigated the in vitro effect of Orn, Hcit and Oro on important parameters of energy metabolism in brain of 30-day-old Wistar rats since mitochondrial abnormalities have been observed in the affected patients. We first verified that Orn and Hcit significantly inhibited the citric acid cycle (inhibition of CO 2 synthesis from [1- 14C] acetate, as well as aconitase and α-ketoglutarate dehydrogenase activities), the aerobic glycolytic pathway (reduced CO 2 production from [U- 14C] glucose) and moderately the electron transfer flow (inhibitory effect on complex I–III). Hcit, but not Orn, was also able to significantly inhibit the mitochondrial creatine kinase activity. Furthermore, this inhibition was prevented by GSH, suggesting a possible role of reactive species oxidizing critical thiol groups of the enzyme. In contrast, the other enzyme activities of the citric acid cycle and of the electron transfer chain, as well as synaptic Na +,K +-ATPase were not altered by either Orn or Hcit at concentrations as high as 5.0 mM. Similarly, Oro did not interfere with any of the tested parameters. Taken together, these data strongly indicate that Orn and Hcit compromise brain energy metabolism homeostasis and Hcit also interferes with cellular ATP transfer and buffering. It is therefore suggested that Orn and especially Hcit may be involved in the neurological damage found in patients affected by HHH syndrome.

α-Ketoglutarate abrogates the nuclear localization of HIF-1α in aluminum-exposed hepatocytes

Aluminum (Al), a known environmental pollutant, has been linked to numerous pathologies such as Alzheimer's disease and anaemia. In this study, we show that α-ketoglutarate (KG) mitigates the Al-mediated nuclear accumulation of hypoxia inducible factor-1α (HIF-1α) in cultured human hepatocytes (HepG2). The nuclear localization of HIF-1α appeared to be triggered by the Al-induced perturbation of prolyl hydroxylase 2 (PHD2). This enzyme was markedly diminished in the Al-challenged hepatocytes. The fate of PHD2 and HIF-1α was intricately linked to the mitochondrial dysfunction observed during Al stress. BN-PAGE, immunoblot, and HPLC revealed that the loss of α-ketoglutarate dehydrogenase (KGDH) and succinate dehydrogenase (SDH) activities were coupled to the accumulation of succinate. However, the treatment of the Al-stressed cells with KG recovered the activity and expression of KGDH, SDH, and PHD2 with a concomitant decrease in the levels of HIF-1α in the nucleus. Taken together, these data indicate that the homeostasis of KG plays a pivotal role in aerobic and anaerobic respiration.

The role of oxidative stress in the development of congestive heart failure in a chicken genotype selected for rapid growth

The present study examined the possible role of reactive oxygen species in the pathogenesis of heart failure in broilers. Data were collected from three groups of birds at various risk of heart failure: Leghorn chickens (resistant to heart failure), slow-growing feed-restricted broilers (low risk of heart failure), fast-growing ad libitum fed broilers (high risk of heart failure), and broilers with congestive heart failure (CHF). In the first part of the study, basic clinical parameters and ultrastructural changes were examined in the context of lipid peroxidation of the ventricular myocardium. This was followed by the study of in vitro changes in the activity of selected cytosolic enzymes (creatine kinase and lactate dehydrogenase) and mitochondrial enzymes (pyruvate dehydrogenase and α-ketoglutarate dehydrogenase) in the presence of oxidants (hydrogen peroxide or tertiary butyl hydroperoxide). The distinctive clinical feature in the fast-growing broilers and in the broilers with CHF as compared with slow-growing broilers or Leghorn chickens was a significantly lower heart rate (P <0.05). Electron microscopy revealed marked morphological changes in myocardial mitochondria in these broilers (i.e. fast-growing broilers and broilers with CHF). The level of malondialdehyde equivalents, an indicator of lipid peroxidation subsequent to generated oxidative stress, was significantly higher (P <0.05) in ad libitum fed broilers and was highest (P <0.01) in broilers with CHF. In vitro, the presence of oxidants had a detrimental effect on creatine kinase and α-ketoglutarate dehydrogenase activity, while lactate dehydrogenase activity increased. The activity of pyruvate dehydrogenase was not altered by oxidants. Our results indicate that the deterioration of heart function in fast-growing commercial broilers in our experimental model is associated with oxidative stress leading to lipid peroxidation of cellular and mitochondrial membranes, and decreased activity of myocardial creatine kinase and α-ketoglutarate dehydrogenase enzymes critical for energy synthesis and transformation pathways.

Synthesis, in vitro and in vivo activity of thiamine antagonist transketolase inhibitors

Tumor cells extensively utilize the pentose phosphate pathway for the synthesis of ribose. Transketolase is a key enzyme in this pathway and has been suggested as a target for inhibition in the treatment of cancer. In a pharmacodynamic study, nude mice with xenografted HCT-116 tumors were dosed with 1 ('N3'-pyridyl thiamine'; 3-(6-methyl-2-amino-pyridin-3-ylmethyl)-5-(2-hydroxy-ethyl)-4-methyl-thiazol-3-ium chloride hydrochloride), an analog of thiamine, the co-factor of transketolase. Transketolase activity was almost completely suppressed in blood, spleen, and tumor cells, but there was little effect on the activity of the other thiamine-utilizing enzymes alpha-ketoglutarate dehydrogenase or glucose-6-phosphate dehydrogenase. Synthesis and SAR of transketolase inhibitors is described.

Mitochondrial dysfunction is responsible for the intestinal calcium absorption inhibition induced by menadione

Menadione (MEN) inhibits intestinal calcium absorption by a mechanism not completely understood. The aim of this work was to find out the role of mitochondria in this inhibitory mechanism. Hence, normal chicks treated with one i.p. dose of MEN were studied in comparison with controls. Intestinal calcium absorption was measured by the in situ ligated intestinal segment technique. GSH, oxidoreductase activities from the Krebs cycle and enzymes of the antioxidant system were measured in isolated mitochondria. Mitochondrial membrane potential was measured by a flow cytometer technique. DNA fragmentation and cytochrome c localization were determined by immunocytochemistry. Data indicate that in 30 min, MEN decreases intestinal Ca2+ absorption, which returns to the control values after 10 h. GSH was only decreased for half an hour, while the activity of malate dehydrogenase and α-ketoglutarate dehydrogenase was diminished for 48 h. Mn2+-superoxide dismutase activity was increased in 30 min, whereas the activity of catalase and glutathione peroxidase remained unaltered. DNA fragmentation and cytochrome c release were maximal in 30 min, but were recovered after 15 h. In conclusion, MEN inhibits intestinal Ca2+ absorption by mitochondrial dysfunction as revealed by GSH depletion and alteration of the permeability triggering the release of cytochrome c and DNA fragmentation.

Activation of ERK1/2 pathway mediates oxidant-induced decreases in mitochondrial function in renal cells

Previously, we showed that oxidant exposure in renal proximal tubular cells (RPTC) induces mitochondrial dysfunction mediated by PKC-epsilon. This study examined the role of ERK1/2 in mitochondrial dysfunction induced by oxidant injury and whether PKC-epsilon mediates its effects on mitochondrial function through the Raf-MEK1/2-ERK1/2 pathway. Sublethal injury produced by tert-butylhydroperoxide (TBHP) resulted in three- to fivefold increase in phosphorylation of ERK1/2 and p38 but not JNK. This was followed by decreases in basal and uncoupled respirations (41%), state 3 respiration and ATP production coupled to complex I (46%), and complex I activity (42%). Oxidant exposure decreased aconitase activity 30% but not pyruvate, alpha-ketoglutarate, and malate dehydrogenase activities. Inhibition of ERK1/2 restored basal and state 3 respirations, DeltaPsi(m), ATP production, and complex I activity but not aconitase activity. In contrast, activation of ERK1/2 by expression of constitutively active MEK1 suppressed basal, uncoupled, and state 3 respirations in noninjured RPTC to the levels observed in TBHP-injured RPTC. MEK1/2 inhibition did not change Akt or p38 phosphorylation, demonstrating that the protective effect of MEK1/2 inhibitor was not due to activation of Akt or inhibition of p38 pathway. Inhibition of PKC-epsilon did not block TBHP-induced ERK1/2 phosphorylation in whole RPTC or in mitochondria. We conclude that 1) oxidant-induced activation of ERK1/2 but not p38 or JNK reduces mitochondrial respiration and ATP production by decreasing complex I activity and substrate oxidation through complex I, 2) citric acid cycle dehydrogenases are not under control of the ERK1/2 pathway in oxidant-injured RPTC, 3) the protective effects of ERK1/2 inhibition are not due to activation of Akt, and 4) ERK1/2 and PKC-epsilon mediate oxidant-induced mitochondrial dysfunction through independent pathways.

Preconditioning prevents loss in mitochondrial function and release of cytochrome c during prolonged cardiac ischemia/reperfusion

Loss in mitochondrial function and induction of mitochondrial-mediated apoptosis occur as a result of cardiac ischemia/reperfusion. Brief and repeated cycles of ischemia/reperfusion, termed ischemic preconditioning, prevent or minimize contractile dysfunction and apoptosis associated with prolonged episodes of cardiac ischemia and reperfusion. The effects of preconditioning on various indices of ischemia/reperfusion-induced alterations in mitochondrial function and structure were therefore explored. Utilizing an in vivo rat model data is provided indicating that preconditioning completely prevents cardiac ischemia/reperfusion-induced: (1) loss in the activity of the redox sensitive Krebs cycle enzyme α-ketoglutarate dehydrogenase; (2) declines in NADH-linked ADP-dependent mitochondrial respiration; (3) insertion of the pro-apoptotic Bcl-2 protein Bax into the mitochondrial membrane; and (4) release of cytochrome c into the cytosol. The results of the current study indicate that preconditioning prevents specific alterations in mitochondrial structure and function that are known to impact cellular viability and provide insight into the collective benefits of preconditioning.

Free radical-mediated neurotoxicity may be caused by inhibition of mitochondrial dehydrogenases in vitro and in vivo

We previously demonstrated that copper facilitated the formation of reactive oxygen species, and inhibited pyruvate dehydrogenase and α-ketoglutarate dehydrogenase in vitro and in animal models of Wilson’s disease in vivo. However, direct Cu 2+ toxicity has only been demonstrated for Wilson’s disease. We now hypothesize that inhibition of these mitochondrial dehydrogenases might also contribute to many other injuries and disorders that are reactive oxygen species-mediated. We have modeled reactive oxygen species-mediated injuries using inducers of reactive oxygen species such as hydrogen peroxide, ethacrynic acid or menadione, or another redox active metal (Cd 2+). Here we demonstrated that these toxic exposures were accompanied by an early marked reduction in both pyruvate dehydrogenase and α-ketoglutarate dehydrogenase activities, followed by a decrease in neuronal mitochondrial transmembrane potential and ATP, prior to murine cortical neuronal death. Thiamine (6 mM), and dihydrolipoic acid (50 μM), required cofactors for pyruvate dehydrogenase and α-ketoglutarate dehydrogenase (thiamine as thiamine pyrophosphate), attenuated the reactive oxygen species-induced reductions in these enzyme activities, as well as subsequent loss of mitochondrial transmembrane potential and ATP, and neuronal death. We next tested the effect of thiamine supplementation on an in vivo model of reactive oxygen species-mediated injury, transient middle cerebral artery occlusion, and reperfusion in rats. Oral or i.p. thiamine administration reduced the middle cerebral artery occlusion-induced infarct. These data suggest that reactive oxygen species-induced neuronal death may be caused in part by reactive oxygen species-mediated inhibition of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase in vitro and in vivo, and that thiamine or dihydrolipoic acid may constitute potential therapeutic agents not just against Cu 2+ neurotoxicity, but may reduce neuronal degeneration in the broader range of diseases mediated by free radical stress.

Disruption of sucA , Which Encodes the E1 Subunit of α-Ketoglutarate Dehydrogenase, Affects the Survival of Nitrosomonas europaea in Stationary Phase

Although Nitrosomonas europaea lacks measurable alpha-ketoglutarate dehydrogenase activity, the recent completion of the genome sequence revealed the presence of the genes encoding the enzyme. A knockout mutation was created in the sucA gene encoding the E1 subunit. Compared to wild-type cells, the mutant strain showed an accelerated loss of ammonia monooxygenase and hydroxylamine oxidoreductase activities upon entering stationary phase. In addition, unlike wild-type cells, the mutant strain showed a marked lag in the ability to resume growth in response to pH adjustments in late stationary phase.

Aconitase is the main functional target of aging in the citric acid cycle of kidney mitochondria from mice

The activities of the citric acid cycle enzymes were determined in mitochondria isolated from kidneys of relatively young, middle age, and old mice. Aconitase exhibited the most significant decrease in activity with age. The activity of α-ketoglutarate dehydrogenase exhibited a modest decrease in activity, while NADP +-isocitrate dehydrogenase (NADP +-ICD) activity increased moderately with age. Activities of citrate synthase, NAD +-isocitrate dehydrogenase (NAD +-ICD), succinyl-CoA synthetase (SCS), succinate dehydrogenase (SD), fumarase (FUM), and malate dehydrogenase (MD) were not affected. The molar ratio of the intra-mitochondrial redox indicator, NADPH:NADP +, was higher in young compared to old animals, while the NADH:NAD + molar ratio remained unchanged. It is suggested that an age-related decrease in aconitase activity along with relatively subtle alterations in activities of some other citric acid cycle enzymes are likely to contribute to a decline in the overall efficiency of mitochondrial bioenergetics. The biological consequences of such alterations include age-related fluctuations in the citric acid cycle intermediates, which are precursors of protein synthesis, activators of fatty acid synthesis, and can also act as ligands for orphan G-protein coupled receptors.