Alpha-ketoglutarate Dehydrogenase Activity Research Articles

Uncovering Actions of Type 3 Deiodinase in the Metabolic Dysfunction-Associated Fatty Liver Disease (MAFLD).

To evaluate the role of D3 in the progression of MAFLD in an animal model. Male/adult Sprague Dawley rats (n = 20) were allocated to a control group (2.93 kcal/g) and high-fat diet group (4.3 kcal/g). Euthanasia took place on the 28th week. D3 activity and expression, Uncoupling Protein 2 (UCP2) and type 1 deiodinase (D1) expression, oxidative stress status, mitochondrial, Krebs cycle and endoplasmic reticulum homeostasis in liver tissue were measured. We observed an increase in D3 activity/expression (p < 0.001) related to increased thiobarbituric acid reactive substances (TBARS) and carbonyls and diminished reduced glutathione (GSH) in the MAFLD group (p < 0.05). There was a D3-dependent decrease in UCP2 expression (p = 0.01), mitochondrial capacity, respiratory activity with increased endoplasmic reticulum stress in the MAFLD group (p < 0.001). Surprisingly, in an environment with lower T3 levels due to high D3 activity, we observed an augmented alpha-ketoglutarate dehydrogenase (KGDH) and glutamate dehydrogenase (GDH) enzymes activity (p < 0.05). Induced D3, triggered by changes in the REDOX state, decreases T3 availability and hepatic mitochondrial capacity. The Krebs cycle enzymes were altered as well as endoplasmic reticulum stress. Taken together, these results shed new light on the role of D3 metabolism in MAFLD.

Protective effect of baicalin on methotrexate-induced mitochondrial damage in testicular tissues of rats

Methotrexate (MTX) is commonly used as a chemotherapeutic drug against cancer but its efficacy is limited due to its side effects. Baicalin is a flavonoid that has potent antioxidant properties. This study aimed to evaluate baicalin’s protective effect on MTX-induced mitochondrial damage in testicular tissues of rats. Twenty-four male Wistar rats were distributed into four groups: control, MTX, MTX + baicalin, and baicalin alone. The activities of enzymes such as catalase, superoxide dismutase, glutathione reductase and glutathione peroxidase was significantly lowered in the MTX treated group, compared to the other groups. Concomitantly, MTX group also showed elevated the levels of reactive oxygen species and thiobarbituric acid reactive substances in the testicular mitochondria. Furthermore, MTX group also showed reduced the activities of tricarboxylic acid (TCA) cycle enzymes, i.e., succinate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, and alpha-ketoglutarate dehydrogenase. The actions of electron transport chain (ETC) complexes (I-IV), as well as mitochondrial membrane potential (MMP), were also reduced the MTX group. However, baicalin treatment reversed the damaging effect of MTX in the mitochondria of testicular tissues. The baicalin treatment potentially reduced the damaging impact of MTX in isolated mitochondria from testicular tissues. Thus, the current study revealed that the baicalin has conspicuous potential to attenuate the MTX induced mitochondrial damage in rat’s testicular tissues.

Abstract 3026: Inhibition of glutaminolysis overcomes metabolic adaptation to devimistat treatment

Abstract The identification and development of reagents that can limit preferential metabolic effectors in tumors as well as trigger unfavorable tumoral metabolic cues is of tremendous importance for suppressing malignancy. However, insufficient blocking of critical metabolic dependencies of cancer allows the development of metabolic bypasses, which significantly dampens therapeutic benefits of selective agents that target a single metabolic enzyme or pathway. We report here that head and neck squamous cell carcinoma (HNSCC) cells display strong addiction to glutamine. Devimistat, a novel lipoate analog, redirects cellular activity towards tumor-promoting glutaminolysis, leading to low anticancer efficacy in HNSCC cells. Mechanistically, devimistatinhibits the tricarboxylic acid cycle by blocking the enzyme activities of pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase, which upregulates GLS1 and eventually promotes the compensatory role of glutaminolysis in cancer cell survival. Most importantly, the addition of a GLS1 inhibitor CB-839 to devimistat treatment abrogates the metabolic dependency of HNSCC cells on glutamine, achieving a synergistic anticancer effect in glutamine-addicted HNSCC. These novel and significant findings could lay a scientific foundation for developing more effective treatments targeting the metabolic requirements of HNSCC. Citation Format: Liwei Lang, Fang Wang, Chloe Shay, Yonggang Ke, Nabil Saba, Yong Teng. Inhibition of glutaminolysis overcomes metabolic adaptation to devimistat treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3026.

Blockade of glutamine-dependent cell survival augments antitumor efficacy of CPI-613 in head and neck cancer

BackgroundAlterations in metabolism are one of the emerging hallmarks of cancer cells and targeting dysregulated cancer metabolism provides a new approach to developing more selective therapeutics. However, insufficient blockade critical metabolic dependencies of cancer allows the development of metabolic bypasses, thus limiting therapeutic benefits.MethodsA series of head and neck squamous cell carcinoma (HNSCC) cell lines and animal models were used to determine the efficacy of CPI-613 and CB-839 when given alone or in combination. Glutaminase 1 (GLS1) depletion was achieved by lentiviral shRNAs. Cell viability and apoptosis were determined in HNSCC cells cultured in 2D culture dish and SeedEZ™ 3D scaffold. Molecular alterations were examined by Western blotting and immunohistochemistry. Metabolic changes were assessed by glucose uptake, lactate production, glutathione levels, and oxygen consumption rate.ResultsWe show here that HNSCC cells display strong addiction to glutamine. CPI-613, a novel lipoate analog, redirects cellular activity towards tumor-promoting glutaminolysis, leading to low anticancer efficacy in HNSCC cells. Mechanistically, CPI-613 inhibits the tricarboxylic acid cycle by blocking the enzyme activities of pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase, which upregulates GLS1 and eventually promotes the compensatory role of glutaminolysis in cancer cell survival. Most importantly, the addition of a GLS1 inhibitor CB-839 to CPI-613 treatment abrogates the metabolic dependency of HNSCC cells on glutamine, achieving a synergistic anticancer effect in glutamine-addicted HNSCC.ConclusionsThese findings uncover the critical role of GLS1-mediated glutaminolysis in CPI-613 treatment and suggest that the CB-839 and CPI-613 combination may potentiate synergistic anticancer activity for HNSCC therapeutic gain.

Protective effects of vitexin on cadmium-induced renal toxicity in rats.

Cadmium (Cd) is an industrial contaminant that poses severe threats to human and animal health. Vitexin (VIT) is a polyphenolic flavonoid of characteristic pharmacological properties. We explored the curative role of vitexin on Cd-induced mitochondrial-dysfunction in rat renal tissues. Twenty-four rats were equally divided into four groups and designated as control, Cd, Cd + vitexin and vitexin treated groups. The results showed that Cd exposure increased urea and creatinine levels while decreased creatinine clearance. Cd reduced the activities of antioxidant enzymes, i.e., catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione content in the Cd exposed group. Cd exposure significantly (p < 0.05) elevated the reactive oxygen species (ROS) and Thiobarbituric acid reactive substances (TBARS) levels in rat kidney. Cd also caused a significant (p < 0.05) reduction in the mitochondrial TCA-cycle enzymes, including isocitrate dehydrogenase, succinate dehydrogenase, alpha-ketoglutarate dehydrogenase, and malate-dehydrogenase activities. Besides, mitochondrial respiratory chain enzymes, including NADH-dehydrogenase, coenzyme Q-cytochrome reductase, succinic-coenzyme Q, and cytochrome c-oxidase activities were also decreased under Cd exposure. Cd exposure also damaged the mitochondrial membrane potential (MMP). However, VIT treatment potentially reduced the detrimental effects of Cd in the kidney of rats. In conclusion, our study indicated that the VIT could attenuate the Cd-induced renal toxicity in rats.

Oxidative stress and hepatocellular mitochondrial dysfunction attenuated by asiatic acid in streptozotocin-induced diabetic rats

Oxidative stress produced by mitochondria is one of the main causes for diabetes associated complications. This study was planned to assess the efficacy of asiatic acid (AA) on oxidative stress mediated hepatocellular mitochondrial dysfunction in streptozotocin (STZ)-induced type-2 diabetic rats. Diabetic rats were generated by intraperitoneal injection of STZ administered as single dose at 40 mg/kg body weight (b. wt.). Then the rats were treated with 20 mg of AA or 600 μg of glibenclamide (Glib)/kg b. wt, once in a day for 45 days. The results of this study illustrated that diabetic rat liver mitochondrial malondialdehyde (MDA), reactive oxygen species (ROS) and protein carbonyl (PCO) levels were significantly increased at the same time antioxidant enzymes and non-enzymes status was markedly diminished when compared with the liver mitochondria of normal control rats. A marked reduction in ROS, MDA, PCO and significant amelioration in non-enzymatic and enzymatic antioxidants were observed in diabetic rats treated with AA or Glib as compared with untreated diabetic control rats. Tricarboxylic acid (TCA) cycle enzymes like isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase and electron transport chain (ETC) complexes activities were significantly diminished in diabetic rats. Besides, mitochondrial uncoupling protein-2 (UCP2) expression was significantly upregulated at the same time adenosine triphosphate (ATP) level and mitochondrial membrane potential (MMP) was markedly lowered in the mitochondria of diabetic rats. AA or Glib treatment with diabetic rats restored the TCA cycle enzymes activities, ETC complexes, UCP2, MMP and ATP level to near normal control rats as compared with untreated diabetic control rats. Hence, the results of the current study indicated that AA ameliorated mitochondrial function by attenuating oxidative stress in STZ-induced type-2 diabetic rats.

Effect of physiological active compounds on status of prooxidative-antioxidative balance and activity of Krebs cycle enzymes in heart and liver tissues under doxorubicin-induced cardiomyopathy

A study was conducted to determinate the effects of Tiotriasolin, Qudesan and the precursors of ubiquinone synthesis (EPM-Mg: alpha-tocopherol acetate), 4-hydroxybenzoic acid, methionine, Mg2+ ions) on the free radical processes and activity of some Krebs cycle dehydrogenases in rat heart and liver tissues under conditions of doxorubicin-induced cardiomyopathy. Administration of doxorubicin intensified the processes of lipid and protein oxidation and suppressed antioxidant enzymes activity in liver and heart tissues. Administration of metabolic correctional drugs caused a decrease the negative effect of doxorubicin on antioxidant system of heart and liver tissues due to increased activity of catalase and superoxide dismutase and inhibition of free radical processes. Doxorubicin administration caused an increase activity of both dehydrogenases in cardiomyocytes, which can be explained by triggering compensatory mechanisms in organ’s cells. The intake of metabolic drugs decreases the negative effect of anthracycline and approximates the dehydrogenase activity, which is close to the control values. In contrast to heart tissues, doxorubicin administration did not cause significant changes in the activity of energy enzymes in liver tissues, which may be due to the presence in the cells of liver a relatively powerful antioxidant defense system (capable in this experimental conditions effectively counteract doxorubicin-induced free radical processes). Administration of Qudesan and EPM-Mg caused a significant increase of alpha-ketoglutarate dehydrogenase and succinate dehydrogenase activities, which indicates the positive effect of these substances on work of the cell energy system.

Manipulation of Amyloid Precursor Protein Processing Impacts Brain Bioenergetics and Glucose Metabolism

The amyloid precursor protein (APP) and one of its terminal cleavage products, amyloid beta (Aβ) are proposed to play a central role in the pathogenesis of Alzheimer's disease (AD). These proteins have also been shown to co-localise within mitochondria with Aβ directly inhibiting the electron transport chain. Cleavage of APP occurs down distinct pathways. Initial cleavage of the full-length protein is mediated by either α- or β-secretase (BACE1). Subsequent cleavage by γ-secretase releases p3 or Aβ protein fragments into the extracellular milieu. Whilst these interactions with mitochondria have been noted, the impact of manipulating APP processing by these distinct pathways on cellular metabolism and bioenergetics has yet to be elucidated. We utilised the human SH-SY5Y neuronal cell line stably overexpressing BACE1 and cortical slices from mice bearing inducible overexpression of APP harbouring the Swedish and Indiana mutations, favouring Aβ production. Protein expression and enzyme activity were determined via western blotting and enzyme-linked immunosorbance assays (ELISA). A Seahorse extracellular flux analyser and radio-labelled substrate assays were used to determine cellular bioenergetics in real-time. All data are expressed as mean ± standard error of the mean and statistical significance determined by Student's t-test. Manipulation of APP cleavage resulted in alterations of 2-deoxyglucose uptake into cells. Chronic elevation in BACE1 resulted in impaired functioning of a key fuel-partitioning enzyme, pyruvate dehydrogenase (PDH, activity reduced to 69 ± 8%, p < 0.05, n=6). This reduced substrate delivery to the mitochondria and increased reliance upon aerobic glycolysis for ATP generation (oxygen consumption rate (OCR) reduced to 65 ± 9%, p < 0.01, n=7 and extracellular acidification rate (ECAR) increased to 165 ± 16%, p < 0.01, n=7). Thus BACE1 overexpression impairs neuronal glucose oxidation resulting in an increased reliance upon aerobic glycolysis for ATP generation. Acute cortical overexpression of APP favouring amyloidogenic processing resulted in a significant reduction in PDH protein expression (reduced to 62 ± 6%, p < 0.01, n = 9-10) and activity of key tricarboxylic acid cycle enzymes, isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase (reduced to 62 ± 7%, p < 0.01, n=6 and 88 ± 2%, p < 0.001, n = 6). Therefore, manipulation of APP processing towards Aβ production in cells and tissue significantly impacts cellular metabolic pathways at the level of glucose uptake, substrate entry to the mitochondria and the TCA cycle.

Oligopeptidi izolovani iz mozga imaju neuroprotektivno dejstvo kod pacova sa sindromom sličnom Parkinsonizmu

Objective. Our research was designed to study the neuroprotective action of the PC-2 peptide complex in rats with experimental Parkinson's disease. Methods. To evaluate the efficacy of the PC-2 peptide complex in rats with experimental Parkinson's disease, levels of 8-isoprostane, noradrenaline, dopamine acetylcholine, glutamate, GABA, aspartate, glycine, as well as the activity of isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase were determined in homogenates of the frontal lobe of the brain. Results. Treatment with the PC-2 peptide complex normalized concentrations of noradrenaline and acetylcholine, increased concentrations of dopamine (however, they did not reach the level in rats from the control group) on the 20th day from the beginning of treatment. The levels of excitatory amino acids in rats with experimental Parkinson's disease increased, while the concentrations of inhibitory amino acids in homogenates of the frontal lobe decreased. The PC-2 protein complex normalized the content of the studied amino acids on the 20th day of treatment. In addition, the content of oxidative stress markers increased in homogenates of the frontal lobe of the brain in rats with Parkinson's disease, which resulted in a decrease in the activity of mitochondrial enzymes and energy formation against the background of high glutamate levels. The normalization of the parameters of energy metabolism was achieved on the 20th day after the surgery. Conclusion. The PC-2 peptide complex regulates the level of biogenic amines and neurotransmitter amino acids and, as a result, affects the energy metabolism in experimental Parkinson's disease.

Abstract 385: D-2-hydroxyglutaric acid Acutely Impairs Oxidative Decarboxylation in the Heart

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1, IDH2) have been described in low-grade glioma and in acute myeloid leukemia. Accumulation of the oncometabolite D-2-hydroxyglutaric acid (2HG) and its release into the blood is associated with dilated cardiomyopathy. The mechanisms leading to changes in cardiac metabolism and contractile function are unknown. We studied in the isolated working rat heart preparation metabolic consequences of increased 2HG supply and its impact on cardiac energy provision. In combination with physiological levels of glucose (5mM) and lactate (0.5mM), hearts were perfused at different concentrations with 2HG (0.5mM, 1.0mM). We confirmed the uptake and enrichment of 2HG in the tissue through liquid chromatography followed by mass spectrometry (LC/MS). 2HG markedly decreased cardiac power and cardiac efficiency in a concentration dependent manner. At the same time glucose oxidation increased significantly (1.4±0.1 μmol/min/g dry wt, p&lt;0.05) at higher workloads. The alpha-Ketoglutarate Dehydrogenase activity was reduced by two-fold (2.84±0.56 μmol/min/g dry wt, p&lt;0.01) and production of reactive oxygen species (e.g. H2O2) increased by three-fold (1.06±0.07 μmol/min/g dry wt, p&lt;0.01) in presence of 2HG. Consistent with this reduction in oxidative decarboxylation, the cytosolic NAD+/NADH redox state increased (3.1±0.1, p&lt;0.001), while the cellular energy charge declined (AMP:ATP ratio, 0.15±0.1, p&lt;0.001). Together, our results demonstrate a direct impairment of cardiac energy substrate metabolism with a resulting decline in contractile function. The data suggests that 2HG directly suppresses cardiac function by specific metabolic alterations in a range of pathologies.

Effects of Strain and Production-Cycle on Indices of Lysine Catabolism in Turkeys

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, Strain A and B, were analyzed 8 times over a period of 17 weeks (n = 8 birds/sampling time/strain) for lysine alpha-ketoglutarate (LKR) and saccharopine dehydrogenase (SDH) activities and mRNA abundance, in vitro lysine oxidation (LOX), L-amino acid oxidase (LAAO) activity, lysyl oxidase (LO) activity and mRNA expression of cationic amino acid transporters (CAT) 1, 2 and 3. We found differences in hepatic LKR activity (p = 0.007), LKR mRNA (p = 0.0004), SDH activity (p = 0.008), LOX (p<0.0001), lysyl oxidase activity (p<0.01), LAAO (p<0.0001) and CAT1 (p = 0.005) and 2 mRNA abundance (p = 0.0022) throughout the production cycle. We also found an effect of strain on SDH (p = 0.046) and CAT-2 mRNA abundance (p = 0.02); while no age or strain effects were detected for CAT3 mRNA abundance. Interestingly, the average LKR and SDH activities across strains and weeks was 240 and 420 nmol per minute per gram of liver, respectively, as opposed to the average LAAO activity, lysyl oxidase activity and L OX which were 0.70, 0.10 and 13.5 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.

Bioenergetic dysfunction in Huntington's disease human cybrids

In this work we studied the mitochondrial-associated metabolic pathways in Huntington's disease (HD) versus control (CTR) cybrids, a cell model in which the contribution of mitochondrial defects from patients is isolated. HD cybrids exhibited an interesting increase in ATP levels, when compared to CTR cybrids. Concomitantly, we observed increased glycolytic rate in HD cybrids, as revealed by increased lactate/pyruvate ratio, which was reverted after inhibition of glycolysis. A decrease in glucose-6-phosphate dehydrogenase activity in HD cybrids further indicated decreased rate of the pentose-phosphate pathway. ATP levels of HD cybrids were significantly decreased under glycolysis inhibition, which was accompanied by a decrease in phosphocreatine. Nevertheless, pyruvate supplementation could not recover HD cybrids' ATP or phosphocreatine levels, suggesting a dysfunction in mitochondrial use of that substrate. Oligomycin also caused a decrease in ATP levels, suggesting a partial support of ATP generation by the mitochondria. Nevertheless, mitochondrial NADH/NAD t levels were decreased in HD cybrids, which was correlated with a decrease in pyruvate dehydrogenase activity and protein expression, suggesting decreased tricarboxylic acid cycle (TCA) input from glycolysis. Interestingly, the activity of alpha-ketoglutarate dehydrogenase, a critical enzyme complex that links the TCA to amino acid synthesis and degradation, was increased in HD cybrids. In accordance, mitochondrial levels of glutamate were increased and alanine was decreased, whereas aspartate and glutamine levels were unchanged in HD cybrids. Conversely, malate dehydrogenase activity from total cell extracts was unchanged in HD cybrids. Our results suggest that inherent dysfunction of mitochondria from HD patients affects cellular bioenergetics in an otherwise functional nuclear background.

Intra- and extra-cellular flux distributions of TCA and glyoxalate cycle and vancomycin production of Amycolatopsis orientalis grown in different glycerol concentration

The relationship between tricarboxylic acid (TCA) and glyoxalate cycle and the effect of their metabolites levels on the vancomycin production of Amycolatopsis orientalis were investigated in different concentration of glycerol (2.5-20 g/l). Intracellular glycerol levels increased with respect to increases in glycerol concentrations of the growth medium. Extracellular glycerol levels decreased slowly up to 24 h while uptake rates were increased during 36-48 h for 10 and 15 g/l and during 36-60 h at 20 g/l of glycerol. Intracellular citrate, alpha-ketoglutarate, fumarate levels increased up to 10 g/l glycerol concentration. However, intracellular succinate and malate levels were increased up to 15 g/l glycerol. Extracellular citrate, alpha-ketoglutarate, succinate and malate levels increased with respect to increases in glycerol concentration. The highest alpha-ketoglutarate dehydrogenase activity was determined at 15 g/l glycerol. Isocitrate lyase activity showed a positive correlation with the increases in glycerol concentration of the growth medium. Vancomycin production increased with the increases in glycerol concentration from 5 to 10 g/l. These results showed that A. orientalis grown in glycerol containing medium used glyoxalate shunt actively instead of TCA cycle which supports precursors of many amino acid which are effective on the antibiotic production.

An Anaerobic-Type α-Ketoglutarate Ferredoxin Oxidoreductase Completes the Oxidative Tricarboxylic Acid Cycle of Mycobacterium tuberculosis

Aerobic organisms have a tricarboxylic acid (TCA) cycle that is functionally distinct from those found in anaerobic organisms. Previous reports indicate that the aerobic pathogen Mycobacterium tuberculosis lacks detectable α-ketoglutarate (KG) dehydrogenase activity and drives a variant TCA cycle in which succinyl-CoA is replaced by succinic semialdehyde. Here, we show that M. tuberculosis expresses a CoA-dependent KG dehydrogenase activity, albeit one that is typically found in anaerobic bacteria. Unlike most enzymes of this family, the M. tuberculosis KG: ferredoxin oxidoreductase (KOR) is extremely stable under aerobic conditions. This activity is absent in a mutant strain deleted for genes encoding a previously uncharacterized oxidoreductase, and this strain is impaired for aerobic growth in the absence of sufficient amounts of CO2. Interestingly, inhibition of the glyoxylate shunt or exclusion of exogenous fatty acids alleviates this growth defect, indicating the presence of an alternate pathway that operates in the absence of β-oxidation. Simultaneous disruption of KOR and the first enzyme of the succinic semialdehyde pathway (KG decarboxylase; KGD) results in strict dependence upon the glyoxylate shunt for growth, demonstrating that KG decarboxylase is also functional in M. tuberculosis intermediary metabolism. These observations demonstrate that unlike most organisms M. tuberculosis utilizes two distinct TCA pathways from KG, one that functions concurrently with β-oxidation (KOR-dependent), and one that functions in the absence of β-oxidation (KGD-dependent). As these pathways are regulated by metabolic cues, we predict that their differential utilization provides an advantage for growth in different environments within the host.

Effects of indole-3-acetic acid on Sinorhizobium meliloti survival and on symbiotic nitrogen fixation and stem dry weight production

We evaluated the effects of the main auxin phytohormone, indole-3-acetic acid (IAA), on the central metabolism of Sinorhizobium meliloti 1021. We either treated S. meliloti 1021 wild-type cells with 0.5 mM IAA, 1021+, or use a derivative, RD64, of the same strain harboring an additional pathway for IAA biosynthesis (converting tryptophan into IAA via indoleacetamide). We assayed the activity of tricarboxylic acid cycle (TCA) key enzymes and found that activity of citrate synthase and alpha-ketoglutarate dehydrogenase were increased in both 1021+ and RD64 as compared to the wild-type strain. We also showed that the intracellular acetyl-CoA content was enhanced in both RD64 and 1021+ strains when compared to the control strain. The activity of key enzymes, utilizing acetyl-CoA for poly-beta-hydroxybutyrate (PHB) biosynthesis, was also induced. The PHB level measured in these cells were significantly higher than that found in control cells. Moreover, 4-week-long survival experiments showed that 80% of 1021 cells died, whereas 50% of RD64 cells were viable. Medicago truncatula plants nodulated by RD64 (Mt-RD64) showed an induction of both acetylene reduction activity and stem dry weight production.

Protective effects of compound FLZ, a novel synthetic analogue of squamosamide, on β-amyloid-induced rat brain mitochondrial dysfunction in vitro

The aim of the present study was to assess the effects of N-[2-(4-hydroxyphenyl)ethyl]-2-(2,5-dimethoxyphenyl)-3-(3-methoxy-4-hydroxyphenyl) acrylamide (compound FLZ), a novel synthetic analogue of squamosamide, on the dysfunction of rat brain mitochondria induced by Abeta(25-35) in vitro. Isolated rat brain mitochondria were incubated with aged Abeta(25-35) for 30 min in the presence and absence of FLZ (1-100 micromol/L). The activities of key mitochondrial enzymes, the production of hydrogen peroxide (H(2)O(2)) and superoxide anion (O2*-), and the levels of glutathione (GSH) in mitochondria were examined. Mitochondrial swelling and the release of cytochrome c from mitochondria were assessed by biochemical and Western blot methods, respectively. Incubation of mitochondria with aged Abeta(25-35) inhibited the activities of alpha-ketoglutarate dehydrogenase (alpha-KGDH), pyruvate dehydrogenase (PDH) and respiratory chain complex IV. It also resulted in increased H(2)O(2) and (O2*-) production, and decreased the GSH level in mitochondria. Furthermore, it induced mitochondrial swelling and cytochrome c release from the mitochondria. The addition of FLZ (100 micromol/L) prior to treatment with Abeta(25-35) significantly prevented these toxic effects of Abeta(25-35) on the mitochondria. FLZ has a protective effect against Abeta(25-35)-induced mitochondrial dysfunction in vitro.

Preventive effect of S-allyl cysteine sulphoxide (Alliin) on mitochondrial dysfunction in normal and isoproterenol induced cardiotoxicity in male Wistar rats: a histopathological study

This study was undertaken to evaluate the preventive role of S-allyl cysteine sulphoxide (SACS) in isoproterenol (ISO)-induced cardiotoxicity in male Wistar rats. Myocardial infarction was induced by subcutaneous injection of ISO (150 mg/kg) once a day for 2 days. SACS (40 and 80 mg/kg) was given as pretreatment orally daily for a period of 35 days using an intragastric tube. SACS pretreatment significantly lowered thiobarbituric acid reactive substances (TBARS) and increased the activities of mitochondrial superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutathione S-transferase (GST), and the concentration of reduced glutathione (GSH) in myocardial infarcted rats. SACS pretreatment also increased significantly the levels of mitochondrial phospholipids and decreased the levels of mitochondrial cholesterol, free fatty acids (FFAs), triglycerides (TGs) and calcium, and the activity of xanthine oxidase (XOD) in heart. Further, the activities of isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH), alpha-ketoglutarate dehydrogenase (alpha-KGDH), NADH-dehydrogenase, and cytochrome C-oxidase were significantly elevated in the mitochondrial fraction of the heart in the SACS-pretreated ISO-induced rats. Oral administration of SACS for a period of 35 days to the normal control rats did not show any significant effect. Histopathological studies of the myocardial tissue showed a protective role of SACS in the myocardial-infarcted rats. The effect at a dose of SACS 80 mg/kg was more effective than the dose 40 mg/kg. The results of the study conclude that SACS protect the mitochondria of the ISO-induced myocardial-infarcted rats.

Ganoderma lucidum (Fr.) P. Karst enhances activities of heart mitochondrial enzymes and respiratory chain complexes in the aged rat

Aging is associated with increased oxidative damage at multiple cellular levels, decline in cellular energy production and enhanced free radical status. The effect of the medicinal mushroom, Ganoderma lucidum on the activities of tricarboxylic acid (Krebs) cycle enzymes and mitochondrial complexes I-IV of the electron transport chain in aged rats were investigated. The activity of Krebs cycle enzymes, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase as well as mitochondrial complexes I, II, III, and IV were determined in heart of aged male Wistar rats orally administrated with 70% ethanolic extract (50 and 250 mg/kg) of G. lucidum. DL-alpha-lipoic acid (100 mg/kg) was taken as the positive control. Administration of the G. lucidum, once daily for 15 days, was significantly (P < 0.05) effective to enhance the Krebs cycle dehydrogenases, and mitochondrial electron transport chain complex IV activities in aged rats. The profound activity of the extract can be correlated to the significant antioxidant property of G. lucidum. The results of the study revealed that G. lucidum is effective to ameliorate the age associated decline of cellular energy status.

Thiamine deficiency-related brain dysfunction in chronic liver failure

End-stage chronic liver failure results in thiamine deficiency caused principally by depletion of liver thiamine stores. Chronic liver failure also leads to increased brain ammonia concentrations. Both ammonia and thiamine deficiency result in decreased activity of alpha-ketoglutarate dehydrogenase, a rate-limiting tricarboxylic acid cycle enzyme. Loss of enzyme activity results in a mitochondrial oxidative deficit in brain and consequent increases in brain lactate, oxidative/nitrosative stress, cellular energy impairment and release of proinflammatory cytokines, all of which have been described in brain in end-stage chronic liver failure. Synergistic effects of ammonia exposure and thiamine deficiency could explain the diencephalic and cerebellar symptomatology described in patients with "hepatic encephalopathy". Unsuspected brain lesions due to thiamine deficiency could explain the incomplete resolution of neuropsychiatric symptoms following the use of ammonia-lowering agents or liver transplantation in patients with end-stage chronic liver failure. These findings underscore the need for prompt, effective thiamine supplementation in all patients with chronic liver failure.

Glutamate Export at the Choroid Plexus in Health, Thiamin Deficiency, and Ethanol Intoxication: Review and Hypothesis

The earliest observed effect in the pathogenesis of experimental Wernicke's encephalopathy and of ethanol intoxication in rats is impairment of the blood cerebrospinal fluid (CSF) barrier at the choroid plexus (CP). For an explanation, these observations direct attention to the role of the CP in maintaining glutamate homeostasis in the CSF. Characteristics of the CP epithelium (CPE) are reviewed, focusing on its role in removal of glutamate from the CSF and its potential for impairment by ethanol oxidation or by thiamin-deficient glucose oxidation. The export of glutamate from CSF to blood at the CP is energy dependent, saturable, and stereospecific. However, the incapacity of the CP to convert glutamate to other metabolites makes it vulnerable to glutamate accumulation should alpha-ketoglutarate dehydrogenase activity be decreased. Elsewhere ethanol metabolism and thiamin-deficiency independently decrease the activity of this mitochondrial enzyme. We argue that they have the same effect within the mitochondria-rich CPE, thereby decreasing energy production necessary for export of glutamate from CSF to blood; diverting its energy metabolism to further glutamate production; and impairing its blood CSF barrier function. This impairment appears to be mediated by glutamate and is attenuated by MK801 but whether it involves one of the CPE glutamate receptors is yet uncertain. This impairment exposes the CSF and hence the paraventricular brain extracellular fluid to neuroactive substances from the blood, including further glutamate, explaining the paraventricular location of neuropathology in Wernicke's encephalopathy. Other organs normally protected from blood by a barrier are affected also by ethanol abuse and by thiamin deficiency, namely the eye, peripheral nerves, and the testis. Much less is known regarding the function of these barriers. Impairment of the CP by ethanol intoxication and by thiamin-deficient carbohydrate metabolism has a common, rational explanation that can guide future research.