Activities Of NADPH-generating Enzymes Research Articles

6-Hydroxy-2,2,4-trimethyl-1,2,3,4-tetrahydroquinoline Demonstrates Neuroprotective Properties in Experimental Parkinson's Disease by Enhancing the Antioxidant System, Normalising Chaperone Activity and Suppressing Apoptosis.

Parkinson's disease (PD) is a neurodegenerative disease, whereby disturbances within the antioxidant defence system, increased aggregation of proteins, and activation of neuronal apoptosis all have a crucial role in the pathogenesis. In this context, exploring the neuroprotective capabilities of compounds that sustain the effectiveness of cellular defence systems in neurodegenerative disorders is worthwhile. During this study, we assessed how 6-hydroxy-2,2,4-trimethyl-1,2,3,4-tetrahydroquinoline (HTHQ), which has antioxidant properties, affects the functioning of the antioxidant system, the activity of NADPH-generating enzymes and chaperones, and the level of apoptotic processes in rats with rotenone-induced PD. Six groups of animals were formed for our experiment, each with 12 animals. These were: a control group, animals with rotenone-induced PD, rats with PD given HTHQ at a dose of 50mg/kg, rats with PD given HTHQ at a dose of 25mg/kg, animals with pathology who were administered a comparison drug rasagiline, and control animals who were administered HTHQ at a dose of 50mg/kg. The study results indicate that administering HTHQ led to a significant decrease in oxidative stress in PD rats. The enhanced redox status in animal tissues was linked with the recovery of antioxidant enzyme activities and NADPH-generating enzyme function, as well as an upsurge in the mRNA expression levels of antioxidant genes and factors Nrf2 and Foxo1. Administering HTHQ to rats with PD normalized the chaperone-like activity and mRNA levels of heat shock protein 70. Rats treated with the compound displayed lower apoptosis intensity when compared to animals with pathology. Therefore, owing to its antioxidant properties, HTHQ demonstrated a beneficial impact on the antioxidant system, resulting in decreased requirements for chaperone activation and the inhibition of apoptosis processes triggered in PD. HTHQ at a dose of 50mg/kg had a greater impact on the majority of the examined variables compared to rasagiline.

SkQ1 improves immune status and normalises the activity of antioxidant and nadph-generating enzymes in adjuvant-induced rheumatoid arthritis in rats

Rheumatoid arthritis (RA) is a severe systemic autoimmune inflammatory disease. Oxidative stress and excessive formation of mitochondrial reactive oxygen species (ROS) are now considered to be the central pathogenetic mechanisms of connective tissue component destruction and the factors responsible for a highly active inflammatory process and autoimmune response. The aim of the present work was to evaluate the effect of mitochondrial-directed antioxidant 10-(6′-plastoquinonyl)decyltriphenylphosphonium (SkQ1) on immune status, intensity of free radical-induced oxidation and functioning of antioxidant system (AOS) and NADPH-generating enzymes in rats with adjuvant-induced RA. Laboratory animals were divided into 4 groups: a control group; a group of animals with RA; animals injected intraperitoneally with SkQ1 at a dose of 1250 nmol/kg every 24 hours during the next 8 days from day 7 of RA development, and animals given SkQ1 at a dose of 625 nmol/kg according to the above scheme. Material for the study was taken on day 15 after the start of the experiment. The rate of erythrocyte sedimentation, the level of circulating immune complexes, and the concentration of immunoglobulins A, M and G were determined in rats by enzyme immunoassay. The intensity of free radical-induced oxidation was assessed by iron-induced biochemiluminescence, diene conjugate content and aconitate hydratase activity. Enzyme activity and metabolite content in animal tissues were analysed spectrophotometrically. The results of the work showed that the development of RA was associated with an increase in the indicators of immune response and intensity of free radical-induced oxidation. The development of an imbalance in AOS functioning and an increase in the activity of NADPH-generating enzymes was also observed. The administration of SkQ1 resulted in a dose-dependent change in the oxidative status indicators towards the control, which was accompanied by the normalization of the immune status parameters. It seems that the tested compound decreased the level of mitochondrial ROS, resulting in an inhibition of the inflammatory response. The consequence of these changes could be inhibition of free radical generation by immunocompetent cells and subsequent mitigation of the severity of oxidative stress in the tissues.

MRNA level of antioxidant genes and activity of NADPH-generating enzymes in rotenone-induced parkinsonism in rats

Aim. To analyze the mRNA level of genes encoding antioxidant enzymes and the transcription factors Nrf2 and Foxo1 regulating their expression and the activity of glucose-6-phosphate dehydrogenase (G6PDH) and NADPdependent isocitrate dehydrogenase (NADP-IDH) and assess the correlation between these parameters, oxidative status, and motor coordination parameters in rats with rotenone-induced parkinsonism.Materials and methods. The study was performed on male Wistar rats aged 4–6 months and weighing 200–250 g. Parkinsonism was modeled by subcutaneous administration of rotenone for 10 days at a dose of 2.5 mg / kg. To confirm the development of the pathology, motor coordination tests and histological staining of the cerebral cortex and striatum with hematoxylin and eosin were used. The oxidative status was analyzed based on the levels of conjugated dienes, carbonyl amino acid residues in proteins, and α-tocopherol. The enzyme activity was studied spectrophotometrically by the formation of NADPH. Real-time PCR was used to analyze the level of gene mRNA.Results. During the study, an increase in serum and brain concentrations of conjugated dienes, carbonyl amino acid residues, and α-tocopherol was observed in the experimental group of rats compared to the controls. It could be associated with the redistribution of this compound between tissues during pathology development. The animals with experimental parkinsonism, in addition, were characterized by a decrease in the mRNA level of the Sod1, Gpx1, Gsr, Gsta2, Nfe2l2, and Foxo1 genes, as well as the activity of G6PDH and NADP-IDH. In the rats with experimental parkinsonism, a negative correlation of NADPH-IDH activity in the brain with serum α-tocopherol level and a positive correlation with Gpx1 and Foxo1 mRNA levels in the striatum were found. The level of oxidatively modified proteins in the brain of the animals with PD was negatively correlated with the concentration of Gsta2 mRNA in the striatum, while the specific activity of G6PDH in the serum was characterized by the positive relationship with grip strength.Conclusion. The data obtained indicate that the inhibition of transcription of the genes encoding antioxidant enzymes and regulatory factors Nrf2 and Foxo1 contributed significantly to the development of oxidative stress in PD. A decrease in the activity of G6PDH and NADP-IDH led to a decrease in the availability of NADPH, which is a limiting factor in the functioning of the glutathione antioxidant system. Obviously, the inhibition of G6PDH and NADP-IDH was also an important pathogenic factor in the progression of the pathology. Along with a decrease in the content of antioxidant gene mRNA in the brain tissues, the level of α-tocopherol increased in the rats with parkinsonism, which could be the result of an imbalance in the functioning of antioxidant system.

The Effect of Combination Therapy with Melatonin on the Enzymes of Glutathione System and the Level of Transforming Growth Factor- β1 in Patients with Type 2 Diabetes Mellitus and Chronic Kidney Disease

Aim. The aim of the work was to assess the effect of combination therapy with melatonin on the clinical and biochemical parameters of chronic kidney disease (CKD) and type 2 diabetes mellitus (DM), the level of transforming growth factor-β1, lipid profile, activity of the glutathione antioxidant system enzymes and the activity of NADPH-generating enzymes in patients.Materials and methods. The study involved 60 people (19 men and 41 women, average age 65.6 ± 9.3 years) with chronic kidney disease associated with type 2 diabetes. The patients were divided into 2 groups. The first group of patients received basic treatment (n = 30, 8 men and 22 women, mean age 64.1 ± 7.9 years); the second group of participants (n = 30, 11 men and 19 women, mean age 69.0 ± 10.5 years) received 2 mg of melatonin in addition to the basic therapy. The control group consisted of 65 apparently healthy individuals (30 men and 35 women, average age 42.3±17.7 years) with normal indicators of general and biochemical blood tests. In the course of the work, the analysis of clinical and biochemical indicators and lipid profile in blood serum, the level of transforming growth factor-β1 by enzyme immunoassay, the activity of enzymes of the glutathione antioxidant system and NADPH-generating enzymes by the spectrophotometric method were carried out.Results. The use of melatonin additionally with basic treatment compared with standard therapy led to a decrease in proteinuria (p=0.010), hyperglycemia (p=0.019), urea concentration (p=0.043), glycated hemoglobin (p=0.045) and transforming growth factor-β1 levels (p=0.020) in patients with CKD. In addition, the use of this drug led to a changing of the lipid profile, and the activity of glutathione antioxidant system enzymes and NADPH-generating enzymes.Conclusion. The differences observed during the study were apparently caused by the action of melatonin, which has nephroprotective and hypoglycemic properties, the ability to neutralize reactive oxygen species and activate the antioxidant system functioning.

Effect of SkQ1 on Activity of the Glutathione System and NADPH-Generating Enzymes in an Experimental Model of Hyperglycemia.

We studied the effect of mitochondria-targeted antioxidant 10-(6'-plastoquinonyl)decyltriphenylphosphonium (SkQ1) on the antioxidant activity of the glutathione system and NADPH-generating enzymes in liver and blood serum of rats with hyperglycemia induced by protamine sulfate. It was found that intraperitoneal injection of SkQ1 prevented both decrease in reduced glutathione level and increase in activity of glutathione system enzymes--glutathione peroxidase, glutathione reductase, and glutathione transferase. Activity of NADPH-generating enzymes--glucose-6-phosphate dehydrogenase and NADP-isocitrate dehydrogenase--was also attenuated by SkQ1. Probably, in this model of hyperglycemia, decreased level of reactive oxygen species in mitochondria led to the decreased burden on the glutathione antioxidant system and NADPH-generating enzymes. Thus, SkQ1 appears to be a promising compound for the treatment and/or prevention of the adverse effects of hyperglycemia.

Methylglyoxal-induced modification of arginine residues decreases the activity of NADPH-generating enzymes

Inadequate control of plasma and cellular glucose and ketone levels in diabetes is associated with increased generation of reactive aldehydes, including methylglyoxal (MGO). These aldehydes react with protein side chains to form advanced glycation end-products (AGEs). Arg residues are particularly susceptible to MGO glycation and are essential for binding NADP+ in several enzymes that generate NADPH, a coenzyme for many critical metabolic and antioxidant enzymes. In most animal cells, NADPH is produced predominantly by glucose-6-phosphate dehydrogenase (G6PD) in the oxidative phase of the pentose phosphate pathway and, to a lesser extent, by isocitrate dehydrogenase (IDH) and malic enzyme (ME). In this study, the activities of isolated G6PD, IDH, and ME were inhibited by MGO (0–2.5mM, 2–3h, 37°C), in a dose- and time-dependent manner, with G6PD and IDH more sensitive to modification than ME. Significant inhibition of these two enzymes occurred with MGO levels ≥500μM. Incubation with radiolabeled MGO (0–500µM, 0–3h, 37°C) demonstrated dose- and time-dependent adduction to G6PD and IDH. HPLC analysis provided evidence for AGE formation and particularly the hydroimidazolones MG-H1 and MG-H2 from Arg residues, with corresponding loss of parent Arg residues. Peptide mass mapping studies confirmed hydroimidazolone formation on multiple peptides in G6PD and IDH, including those critical for NADP+ binding, and substrate binding, in the case of IDH. These results suggest that modification of NADPH-producing enzymes by reactive aldehydes may result in alterations to the cellular redox environment, potentially predisposing cells to further damage by oxidants and reactive aldehydes.

Effect of Early Feed Restriction on Metabolic Programming and Compensatory Growth in Broiler Chickens

The effect of early feed restriction on metabolic programming and compensatory growth was studied in broiler chickens. A total of 480 female 1-d-old broiler birds (Aconred) were randomly allocated to ad libitum and feed-restricted groups, each of which was replicated 6 times with 40 birds per replicate. Broilers were provided commercial diets. Feed-restricted broilers were deprived of feed for 4 h per day from 1 to 21 d of age. Effects of treatments were determined at 21 and 63 d of age. In feed-restricted birds at 21 d of age, BW, average daily gain and average daily feed intake, breast muscle (P < 0.01), carcass yield (P < 0.05), and abdominal fat (P < 0.05) were decreased. Ether extract content in breast muscle was increased (P < 0.01), whereas CP content was slightly decreased. Triiodothyronine (P < 0.01) and thyroxine (P < 0.05) were decreased in serum. Free fatty acid and very low density lipoprotein were slightly increased in serum, whereas triglyceride and glucose were decreased (P < 0.01). Activities of NADPH-generating enzymes in liver including malic dehydrogenase, isocitrate dehydrogenase, and glucose-6-phosphate remained unchanged in ad libitum birds, whereas hormone-sensitive lipase activity was increased (P < 0.01). In feed-restricted birds at 63 d of age, BW, average daily gain, average daily feed intake, carcass yield, breast muscle yield, and serum triiodothyronine and thyroxine remained as ad libitum birds, whereas abdominal fat yield was increased (P < 0.05). Ether extract content in breast muscle was decreased (P < 0.01), whereas CP content was increased (P < 0.05). Activities of NADPH-generating enzymes were significantly increased, except abdominal malic dehydrogenase and hormone-sensitive lipase activity was decreased (P < 0.01) in liver and abdominal fat. Lipoprotein lipase activity was increased (P < 0.05) in abdominal fat. In summary, feed restriction severely affected growth performance and lipid metabolism in broilers in the early period. Because there was no statistical difference among the final BW, near full compensatory growth was achieved. In addition, early feed restriction might have induced prolonged metabolic programming in chicks and led to adult obesity.

Glutathione System and Activity of NADPH-Generating Enzymes in the Liver of Intact Rats and Animals with Toxic Hepatitis Receiving Melatonin

Melatonin administered to intact animals increased glutathione concentration and activities of glutathione peroxidase, glutathione reductase, NADP-isocitrate dehydrogenase, and glucose-6-phosphate dehydrogenase in the liver. In animals with toxic hepatitis melatonin treatment decreased glutathione concentration and enzyme activities, which was probably associated with inhibition of free radical oxidation under the influence of this hormone.

Peroxisome proliferator perfluorodecanoic acid alters glutathione and related enzymes.

Previously we have shown that treatment with the peroxisome proliferator perfluorodecanoic acid (PFDA) significantly increased hepatic reduced glutathione (GSH) content without altering the activity of selenium-glutathione peroxidase. In this study we examined some potential mechanisms by which PFDA treatment increases GSH levels. Male Sprague-Dawley rats were given a single injection of 0, 8.8, 17.5, and 35 mg PFDA in corn oil per kg body weight. Twelve days later the effects of PFDA on the activities of enzymes associated with GSH synthesis, utilization, and regeneration were assessed. The results showed that in a dose-dependent manner, PFDA treatment significantly decreased the activity of gamma-glutamylcysteine synthetase, while the activities of NADPH-generating enzymes, malic enzyme, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase were increased. PFDA treatment also dose dependently decreased cytosolic, but not microsomal, glutathione S-transferase activity, and the activity of glutathione reductase was decreased by the highest dose of PFDA. The data obtained suggest that increased hepatic GSH levels following PFDA treatment may result from increased regeneration and/or decreased utilization.

Effect of dietary sodium phytate on the hepatic and serum levels of lipids and on the hepatic activities of NADPH-generating enzymes in rats fed on sucrose.

The effect was investigated of dietary 0.5% sodium phytate on the hepatic and serum lipid status in rats fed on sucrose for 29 or 30 days. The increase in hepatic weight, levels of hepatic total lipids, triglyceride, cholesterol, serum triglyceride and phospholipid, and the activities of hepatic NADPH-generating enzymes due to sucrose feeding were generally depressed by dietary sodium phytate.

Dietary fat effects on hepatic lipid peroxidation and enzymes of H2O2 metabolism and NADPH generation.

The purpose of this study was to determine the effects of dietary fat quantity and fatty acid composition on hepatic H2O2-metabolizing systems, activities of NADPH-generating enzymes and lipid peroxidation. One-month-old male C57BL/6J mice were fed one of six diets: (i) 5% fat, rich in 18:2n-6 fatty acid (5% N-6); (ii) 20% fat, rich in 18:3n-3 (N-3); (iii) 20% fat, rich in 18:2n-6 (N-6); (iv) 20% fat, rich in 18:1n-9 (N-9); (v) 20% fat, rich in saturated fatty acids (SAT); and (vi) 20% fat, deficient in essential fatty acids (EFAD); for 11 wk. Comparisons between animal groups receiving different fat quantities showed that activities of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) and malic enzyme (ME, EC 1.1.1.40) and the levels of conjugated dienes were significantly lower in the N-6 than in 5% N-6 group. Conversely, activities of catalase (CAT, EC 1.11.1.6) and selenium-glutathione peroxidase (SeGSHPx, EC 1.11.1.9) were higher in the N-6 than in 5% N-6 group. Among the five dietary groups receiving 20% fat but differing in fatty acid composition, CAT activity was lower in the N-9 group, SeGSHPx activity was lower in the EFAD group, and glutathione reductase (GSSGR, EC 1.6.4.2) activity was higher in the N-6 than in the N-3, N-9, SAT and EFAD group. The EFAD group had much higher levels of total lipids and conjugated dienes, as well as activities of NADPH-generating enzymes, including G6PDH, ME and isocitrate dehydrogenase (EC 1.1.1.42), than the other four high-fat groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Iron Reduction and Trans Plasma Membrane Electron Transfer in the Yeast Saccharomyces cerevisiae

The ferri-reductase activity of whole cells of Saccharomyces cerevisiae (washed free from the growth medium) was markedly increased 3 to 6 h after transferring the cells from a complete growth medium (preculture) to an iron-deficient growth medium (culture). This increase was prevented by the presence of iron, copper, excess oxygen, or other oxidative agents in the culture medium. The cells with increased ferri-reductase activity had a higher reduced glutathione content and a higher capacity to expose exofacial sulfhydryl groups. Plasma membranes purified from those cells exhibited a higher reduced nicotinamide adenine phosphate (NADPH)-dependent ferri-reductase specific activity. However, the intracellular levels of NADPH, NADH, and certain organic acids of the tricarboxylic acids cycle were unchanged, and the activity of NADPH-generating enzymes was not increased. Addition of Fe(III)-EDTA to iron-deprived and iron-rich cells in resting suspension resulted in a decrease in intracellular reduced glutathione in the case of iron-deprived cells and in an increase in organic acids and a sudden oxidation of NADH in both types of cells. The depolarizing effect of Fe(3+) was more pronounced in iron-rich cells. The metabolic pathways that may be involved in regulating the trans-plasma membrane electron transfer in yeast are discussed.

Relationships between blood groups, isozymes and halothane reaction in pigs from a selection experiment.

German Landrace pigs (n = 1500) were halothane-tested and blood samples were taken for the determination of A-O and H blood types as well as for the determination of PHI and 6-PGD isozymes. The pigs originated from two generations (7th and 8th) of a selection experiment 'selection for activity of NADPH-generating enzymes in backfat of pigs'. The selection lines are E-, E+ (selection for low and high enzyme activity), U- (selection for low ultrasonic backfat thickness) and K (control). Preliminary results show an average proportion of halothane-susceptible animals of 49%. The frequencies of halothane-positive pigs amount to 60%, 46%, 70% and 30% in lines E-, E+, U- and K, respectively. The investigation shows a non-random combination of the marker genes caused by linkage disequilibrium, especially in line E-. Recombination frequencies between the loci vary from 0% to 18%.

Changes in activity of enzymes related to glycolysis, gluconeogenesis and lipogenesis in placentae from diabetic women

The activity of enzymes with a regulatory function in the pathways of glycolysis, gluconeogenesis and NADP-generation was investigated in 50 placentae from normal pregnancies and deliveries, 23 placentae from women with gestational diabetes, and 12 placentae from insulin-dependent patients. In placentae from the gestational diabetic group, the activity of pyruvate kinase and of NADPH-generating enzymes was raised and the activity of enzymes connected to glucogenesis was unchanged. These alterations were attributed to the oversupply of glucose and insulin to morphologically normal and well-oxygenated placental tissue. In the placentae from the insulin-dependent group, the activity of pyruvate kinase was reduced, the activity of NADPH-generating enzymes was enhanced and the activity of those related to the gluconeogenesis was unchanged. It is suggested that this pattern of enzyme changes reflects a reduction in the glycolytic capacity in these placentae, which may be due to inhibition by products of enhanced fatty acid oxidation in diabetes, amino acids and/or by long-term anoxia as a result of uteroplacental circulatory disturbances. The possible relation of reduced energy-forming capacity of the placenta in diabetes to its transport function is discussed.

Alterations of NADPH-Generating and Drug-Metabolizing Enzymes by Feed Restriction in Male Rats

Two groups of weanling male Sprague-Dawley rats were fed a semisynthetic diet (ad libitum group) and 50% of that consumed by the ad libitum group (restricted group) for 49 days. Compared to the ad libitum group, the animals of the restricted group gained 55% less in body weight and had 60% smaller livers. While serum glucose, urea nitrogen, uric acid and lipids were lowered in the restricted animals, other serum biochemical parameters remained unchanged. The in vitro activities of hepatic drug-metabolizing enzymes were increased two- to threefold in the restricted animals. Feed restriction also caused a significant increase in the activities of NADPH-generating enzymes of liver and adipose tissue; however, the enzymes not concerned with the production of NADPH remained unaffected by feed restriction. It is concluded that feed restriction, unlike starvation, enhanced activities of drug-metabolizing enzymes as well as NADPH-generating enzymes.feed restriction drug-metabolizing enzymes NADPH-generating enzymes lipids fatty acids

Fatty Acid Synthesis from Lactate in Growing Cattle

Rates of fatty acid synthesis from lactate and acetate and activities of selected lipogenic and NADPH-generating enzymes were determined in subcutaneous, intermuscular and intramuscular adipose tissues of cattle that were 11–19 months of age. Fatty acid synthesis from lactate and acetate increased from 11 to 13 months of age in subcutaneous and intermuscular adipose tissues; synthesis from lactate increased until 17 months of age. In intramuscular adipose tissue, synthesis from lactate also increased until 17 months of age while that from acetate continually increased. Activities of NADPH-generating enzymes increased in all three fat depots from 11 to 13 months in age, and little change occurred thereafter. Acetyl-CoA carboxylase activity was constant over entire growth period in all depots. Activity of ATP-citrate lyase increased from 11 to 13 months of age in subcutaneous and intermuscular adipose tissues, but did not increase until 19 months of age in intramuscular adipose tissue. In all cases, activities of ATP-citrate lyase were sufficient to support synthesis from lactate; therefore, lactate conversion to fatty acids in bovine adipose tissues seems to use the citrate cleavage pathway for generation of cytosolic acetyl-CoA.lactate fatty acid synthesis cattle citrate cleavage pathway

Phenotypic and genetic parameters of the activities of NADPH-generating enzymes in swine adipose tissue

Phenotypic and genetic parameters of the activities of NADPH-generating enzymes in swine adipose tissue

Influence of temperature on the composition of fatty acids and on lipogenesis in frog tissues

1. 1. Frogs ( Rana esculenta) were adapted to various temperatures and the fatty acid composition of tissues and lipogenesis from radioactive precursors were studied. The activities of NADPH-generating enzyme systems were also compared. 2. 2. Liver and adipose tissue from cold-adapted frogs contained more unsaturated fatty acids and less saturated, than the corresponding tissues from frogs kept at higher temperatures. However, differences were found mostly in the proportions of polyenoic acids and not in those of the monoenoic acids. The changes in fatty acid composition of phospholipids and of neutral lipids were similar. 3. 3. As a result of in vitro lipogenesis from 1-C 14-acetate mostly saturated and monoenoic fatty acids were formed and there was no indication of de novo formation of linoleic acid in tissues from either cold-adapted frogs or those kept at 20°C. 4. 4. The temperature of adaptation did not influence either the extent of hepatic lipogenesis from C 14-acetate or the distribution of radioactivity in lipid fractions. On the other hand, in adipose tissue of frogs kept at 20°C both lipogenesis and esterification of newly formed fatty acids were strongly inhibited as compared with the tissue from cold-adapted animals. 5. 5. No correlation between the rate of lipogenesis and the activities of NADPH-generating enzymes could be observed and under the conditions of depressed lipogenesis formation of NADPH did not seem to be rate limiting.