Mouse-liver glutathione reductase. Purification, kinetics, and regulation.

Glutathione reductase from the liver of DBA/2J mice was purified to homogeneity by means of ammonium sulfate fractionation and two subsequent affinity chromatography steps using 8-(6-aminohexyl)-amino-2'-phospho-adenosine diphosphoribose and N6-(6-aminohexyl)-adenosine 2',5'-biphosphate-Sephadex columns. A facile procedure for the synthesis of 8-(6-aminohexyl)-amino-2'-phospho-adenosine diphosphoribose is also presented. The purified enzyme exhibits a specific activity of 158 U/mg and an A280/A460 of 6.8. It was shown to be a dimer of Mr 105000 with a Stokes radius of 4.18 nm and an isoelectric point of 6.46. Amino acid composition revealed some similarity between the mouse and the human enzyme. Antibodies against mouse glutathione reductase were raised in rabbits and exhibited high specificity. The catalytic properties of mouse liver glutathione reductase have been studied under a variety of experimental conditions. As with the same enzyme from other sources, the kinetic data are consistent with a 'branched' mechanism. The enzyme was stabilized against thermal inactivation at 80 degrees C by GSSG and less markedly by NADP+ and GSH, but not by NADPH or FAD. Incubation of mouse glutathione reductase in the presence of NADPH or NADH, but not NADP+ or NAD+, produced an almost complete inactivation. The inactivation by NADPH was time, pH and concentration dependent. Oxidized glutathione protected the enzyme against inactivation, which could also be reversed by GSSG or other electron acceptors. The enzyme remained in the inactive state even after eliminating the excess NADPH. The inactive enzyme showed the same molecular weight as the active glutathione reductase. The spectral properties of the inactive enzyme have also been studied. It is proposed that auto-inactivation of glutathione reductase by NADPH and the protection as well as reactivation by GSSG play in vivo an important regulatory role.     

Properties and physiological function of a glutathione reductase purified from spinach leaves by affinity chromatography

Glutathione reductase (EC 1.6.4.2) was purified from spinach (Spinacia oleracea L.) leaves by affinity chromatography on ADP-Sepharose. The purified enzyme has a specific activity of 246 enzyme units/mg protein and is homogeneous by the criterion of polyacrylamide gel electrophoresis on native and SDS-gels. The enzyme has a molecular weight of 145,000 and consists of two subunits of similar size. The pH optimum of spinach glutathione reductase is 8.5–9.0, which is related to the function it performs in the chloroplast stroma. It is specific for oxidised glutathione (GSSG) but shows a low activity with NADH as electron donor. The pH optimum for NADH-dependent GSSG reduction is lower than that for NADPH-dependent reduction. The enzyme has a low affinity for reduced glutathione (GSH) and for NADP⁺, but GSH-dependent NADP⁺ reduction is stimulated by addition of dithiothreitol. Spinach glutathione reductase is inhibited on incubation with reagents that react with thiol groups, or with heavymetal ions such as Zn²⁺. GSSG protects the enzyme against inhibition but NADPH does not. Pre-incubation of the enzyme with NADPH decreases its activity, so kinetic studies were performed in which the reaction was initiated by adding NADPH or enzyme. The Km for GSSG was approximately 200 M and that for NADPH was about 3 M. NADP⁺ inhibited the enzyme, assayed in the direction of GSSG reduction, competitively with respect to NADPH and non-competitively with respect to GSSG. In contrast, GSH inhibited non-competitively with respect to both NADPH and GSSG. Illuminated chloroplasts, or chloroplasts kept in the dark, contain equal activities of glutathione reductase. The kinetic properties of the enzyme (listed above) suggest that GSH/GSSG ratios in chloroplasts will be very high under both light and dark conditions. This prediction was confirmed experimentally. GSH or GSSG play no part in the light-induced activation of chloroplast fructose diphosphatase or NADP⁺-glyceraldehyde-3-phosphate dehydrogenase. We suggest that GSH helps to stabilise chloroplast enzymes and may also play a role in removing H₂O₂. Glucose-6-phosphate dehydrogenase activity may be required in chloroplasts in the dark in order to provide NADPH for glutathione reductase.Abbreviations GSH reduced form of the tripeptide glutathione - GSSG oxidised form of glutathione     

Cyclic strain modulates resistance to oxidant stress by increasing G6PDH expression in smooth muscle cells

Vascular smooth muscle cells (VSMC) may be subjected to mechanical forces, such as cyclic strain, that promote the formation of reactive oxygen species (ROS). We hypothesized that VSMC modulate this adverse milieu by increasing the expression of glucose-6-phosphate dehydrogenase (G6PDH) to maintain or restore intracellular glutathione (GSH) levels. Cyclic strain increased superoxide formation, which resulted in diminished GSH because of an increase in oxidized glutathione formation; there was also an increase in glutathione peroxidase and glutathione reductase activities. G6PDH activity and protein expression were enhanced concomitant with decreases in GSH levels and remained elevated until intracellular GSH levels were restored. To confirm the role of G6PDH in repleting GSH stores, we inhibited G6PDH activity with DHEA or inhibited enzyme expression with an antisense oligodeoxynucleotide. Diminished G6PDH activity or expression was associated with persistently depleted GSH levels and inhibition of the cyclic strain-mediated increase in glutathione reductase activity. These observations demonstrate that cyclic strain promotes oxidant stress in VSMC, which, in turn, induces G6PDH expression. When G6PDH is inhibited, GSH levels are not restored because of impaired glutathione reductase activity. These data suggest that G6PDH is a critical determinant of the response to oxidant stress in VSMC.     

In Vivo and in Vitro Studies of Glucose-6-Phosphate Dehydrogenase from Barley Root Plastids in Relation to Reductant Supply for NO2- Assimilation

Pyridine nucleotide pools were measured in intact plastids from roots of barley (Hordeum vulgare L.) during the onset of NO2- assimilation and compared with the in vitro effect of the NADPH/NADP ratio on the activity of plastidic glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) from N-sufficient or N-starved roots. The NADPH/NADP ratio increased from 0.9 to 2.0 when 10 mM glucose-6-phosphate was supplied to intact plastids. The subsequent addition of 1 mM NaNO2 caused a rapid decline in this ratio to 1.5. In vitro, a ratio of 1.5 inactivated barley root plastid G6PDH by approximately 50%, suggesting that G6PDH could remain active during NO2- assimilation even at the high NADPH/NADP ratios that would favor a reduction of ferredoxin, the electron donor of NO2- reductase. Root plastid G6PDH was sensitive to reductive inhibition by dithiothreitol (DTT), but even at 50 mM DTT the enzyme remained more than 35% active. In root plastids from barley starved of N for 3 d, G6PDH had a substantially reduced specific activity, had a lower Km for NADP, and was less inhibited by DTT than the enzyme from N-sufficient root plastids, indicating that there was some effect of N starvation on the G6PDH activity in barley root plastids.     

Effects of Exogenous Proline and Glycinebetaine on the Salt Tolerance of Rice Cultivars

Glutathione reductase was purified from iron-grown Thiobacillus ferrooxidas AP19-3 to an electrophoretically homogeneous state. The enzyme had an apparent molecular weight of 100,000 and was composed of two identical subunits of molecular weight (M_rs, 52,000) as estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. A purified enzyme reduced one mole of the oxidized form of glutathione (GSSG) with one mole of NADPH to produce two moles of the reduced form of glutathione (GSH) and one mole of NADP⁺. The glutathione reductase was most active at pH 6.5 and 40°C, and had an isoelectric point at 5.1. The Michaelis constants of glutathione reductase for GSSG, NADPH, and NADH were 300, 26, and 125 μM, respectively.     

Cold-hardiness-specific glutathione reductase isozymes in red spruce. Thermal dependence of kinetic parameters and possible regulatory mechanisms.

The thermal dependence of kinetic parameters has been determined in purified or partially purified preparations of cold-hardiness-specific glutathione reductase isozymes from red spruce (Picea rubens Sarg.) needles to investigate a possible functional adaptation of these isozymes to environmental temperature. We have previously purified glutathione reductase isozymes specific for nonhardened (GR-1NH) or hardened (GR-1H) needles. Isozymes that were distinct from GR-1NH and GR-1H, but appeared to be very similar to each other, were also purified from nonhardened (GR-2NH) or hardened (GR-2H) needles (A. Hausladen, R.G. Alscher [1994] Plant Physiol 105: 205-213). GR-1NH had 2-fold higher Km values for NADPH and 2- to 4-fold lower Km values for oxidized glutathione (GSSG) than GR-2NH, and a similar difference was found between GR-1H and GR-2H. However, no differences in Km values were found between the hardiness-specific isozymes GR-1NH and GR-1H. There was only a small effect of temperature on the Km(GSSG) of GR-1H and GR-2H, and no significant temperature effect on Km(NADPH) or Km(GSSG) could be found for the other isozymes. These results are discussed with respect to "thermal kinetic windows," and it is proposed that the relative independence of Km values to temperature ensures adequate enzyme function in a species that is exposed to extreme temperature differences in its natural habitat. A variety of substrates has been tested to characterize any further differences among the isozymes, but all isozymes are highly specific for their substrates, NADPH and GSSG. The reversible reductive inactivation by NADPH (redox interconversion) is more pronounced in GR-1H than in GR-2H. Reduced, partially inactive GR-1H is further deactivated by H2O2, whereas GR-2H is fully reactivated by the same treatment. Both isozymes are reactivated by GSSG or reduced glutathione. It is proposed that this property of GR-2H ensures enzyme function under oxidative conditions, and that in vivo the enzyme may exist in its partially inactive form and be activated in the presence of increased levels of GSSG or oxidants.     

Blood glutathione redox status in gestational hypertension

Gestational hypertension during the third trimester reflects an exaggerated maternal inflammatory response to pregnancy. We hypothesized that oxidative stress present even in normal pregnancy becomes uncompensated in hypertensive patients. A glucose-6-phosphate dehydrogenase (G6PD) activity sufficient to meet the increased reductive equivalent need of the cells is indispensable for defense against oxidative stress. The erythrocyte glutathione redox system was studied, where G6PD is the only NADPH source. The glutathione (GSH) redox status was measured both in vivo and after an in vitro oxidative challenge in pregnant women with gestational hypertension (n = 19) vs. normotensive pregnant subjects (n = 18) and controls (n = 20). An erythrocyte GSH depletion with an increase in the oxidized form (GSSG) resulted in an elevated ratio GSSG/GSH (0.305 +/- 0.057; mean +/- SD) in hypertensive pregnant women vs. normotensive pregnant or control subjects (0.154 +/- 0.025; 0.168 +/- 0.073; p <.001). In hypertensive pregnant patients, a "GSH stability" decrease after an in vitro oxidative challenge suggested a reduced GSH recycling capacity resulting from an insufficient NADPH supply. The erythrocyte GSSG/GSH ratio may serve as an early and sensitive parameter of the oxidative imbalance and a relevant target for future clinical trials to control the effects of antioxidant treatment in women at increased risk of the pre-eclampsia syndrome.     

Sulfate reduction in Catharanthus roseus (L.)

Cell homogenates from Catharanthus roseus (L.) G. Don. grown S-autotrophically on sulfate in the dark are capable of reducing adenylysulfate (APS) to cysteine. This reduction required a particulate protein fraction from the cell extract and reduced ferredoxin as the electron donor. The protein fraction (MW 700,000±50,000) was found to contain Fd:NADP⁺ reductase, glutathione reductase and an unspecific dithiol reductase, and APS-sulfotransferase and thiosulfonate reductase activity. Resolution into these individual enzyme activities led to a non-restorable loss of the APS reducing activity. It was observed that a slow gradual decay of the APS reducing activity was accompanied by a likewise slow generation of a ferredoxin-dependent sulfite reductase.Enzymes and abbreviations APS Adenosine 5-phosphosulfate - APS-kinase E.C.2.7.1.25 - ATP-sulfurylase E.C.2.7.7.4 - Fd ferredoxin - Fd-NADP⁺-reductase E.C.1.6.7.1. - Glutathione reductase E.C.1.6.4.2. - G6P Glucose 6-phosphate - G6PDH glucose 6-phosphate dehydrogenase, E.C.1.1.49 - GSSG oxidized glutathione - GSSO₃H S-sulfoglutathione - MVH reduced methylviologen - OASS O-acetylserine sulfhydrylase-E.C. 4.2.99.8 - Sulfite reductase E.C.1.8.1.2     

Antioxidant defences of the apoplast

Summary The apoplast of barley and oat leaves contained superoxide dismutase (SOD), catalase, ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, and glutathione reductase activities. The activities of these enzymes in the apoplastic extracts were greatly modified 24 h after inoculation with the biotrophic fungal pathogenBlumeria graminis. The quantum efficiency of photosystem II, which is related to photosynthetic electron transport flux, was comparable in inoculated and healthy leaves during this period. Apoplastic soluble acid invertase activity was also modified in inoculated leaves. Inoculation-dependent increases in apoplastic SOD activity were observed in all lines. Major bands of SOD activity, observed in apoplastic protein extracts by activity staining of gels following isoelectric focusing, were similar to those observed in whole leaves but two additional minor bands were found in the apoplastic fraction. The apoplastic extracts contained substantial amounts of dehydroascorbate (DHA) but little or no glutathione (GSH). Biotic stress decreased apoplastic ascorbate and DHA but increased apoplastic GSH in resistant lines. The antioxidant cycle enzymes may function to remove apoplastic H₂O₂ with ascorbate and GSH derived from the cytoplasm. DHA and oxidized glutathione may be reduced in the apoplast or returned to the cytosol for rereduction.Abbreviations AA reduced ascorbate - APX ascorbate peroxidase - DHA dehydroascorbate (oxidised ascorbate) - DHAR dehydroascorbate reductase - G6PDH glucose-6-phosphate dehydrogenase - GSH reduced glutathione - GSSG glutathione disulphide - GR glutathione reductase - MDHA monodehydroascorbate - MDHAR monodehydroascorbate reductase - SOD superoxide dismutase     

Modification of microsomal 11beta-HSD1 activity by cytosolic compounds: glutathione and hexose phosphoesters

11beta-Hydroxysteroid dehydrogenase1(11beta-HSD1) can serve either as an oxo-reductase or dehydrogenase determined by the redox state in the endoplasmic reticulum (ER). This bidirectional enzyme governs paracrine glucocorticoid production. Recent in vitro studies have underscored the key role of cytoplasmic glucose-6-phosphate (G6P) in controlling the flux direction of 11betaHSD-1 by altering the intraluminal ER NADPH/NADP ratio. The hypothesis that other hexose phosphoesters or the plentiful cellular oxidative protector glutathione could also regulate microsomal 11betaHSD-1 activity was tested. Fructose-6-phosphate increased the activity of 11beta-HSD1 reductase in isolated rat and porcine liver microsomes but not porcine fat microsomes. Moreover, oxidized glutathione (GSSG) attenuated 11beta-HSD1 reductase activity by 40% while reduced glutathione (GSH) activated the reductase in liver. Fat microsomes were unaffected because they lack glutathione reductase. Nonetheless, another oxidizing agent, hydrogen peroxide (0.5mM), inhibited both fat and liver 11beta-HSD1 reductase. Consistent with the major role of the redox state, 2.5mM GSSG and hydrogen peroxide augmented the 11beta-HSD1 dehydrogenase, antithetical to the reductase, by 20-30% in liver microsomes. Given the key role of reactive oxygen species and hexose phosphate accumulation in the pathoetiology of obesity and diabetes, these compounds might also modify 11beta-HSD1 in these conditions.     

Nitrite reduction in barley-root plastids: Dependence on NADPH coupled with glucose-6-phosphate and 6-phosphogluconate dehydrogenases,and possible involvement of an electron carrier and a diaphorase

Plastids from roots of barley (Hordeum vulgare L.) seedlings were isolated by discontinuous Percoll-gradient centrifugation. Coinciding with the peak of nitrite reductase (NiR; EC 1.7.7.1, a marker enzyme for plastids) in the gradients was a peak of a glucose-6-phosphate (Glc6P) and NADP⁺-linked nitrite-reductase system. High activities of phosphohexose isomerase (EC 5.3.1.9) and phosphoglucomutase (EC 2.7.5.1) as well as glucose-6-phosphate dehydrogenase (Glc6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) were also present in the isolated plastids. Thus, the plastids contained an overall electron-transport system from NADPH coupled with Glc6PDH and 6PGDH to nitrite, from which ammonium is formed stoichiometrically. However, NADPH alone did not serve as an electron donor for nitrite reduction, although NADPH with Glc6P added was effective. Benzyl and methyl viologens were enzymatically reduced by plastid extract in the presence of Glc6P+ NADP⁺. When the plastids were incubated with dithionite, nitrite reduction took place, and ammonium was formed stoichiometrically. The results indicate that both an electron carrier and a diaphorase having ferredoxin-NADP⁺ reductase activity are involved in the electron-transport system of root plastids from NADPH, coupled with Glc6PDH and 6PGDH, to nitrite.Abbreviations Cyt cytochrome - Glc6P glucose-6-phosphate - Glc6PDH glucose-6-phosphate dehydrogenase - MVH reduced methyl viologen - NiR nitrite reductase - 6PG 6-phosphogluconate - 6PGDH 6-phosphogluconate dehydrogenase     

Glucose-6-phosphate dehydrogenase plays a central role in modulating reduced glutathione levels in reed callus under salt stress

In the present study, we investigated the role of glucose-6-phosphate dehydrogenase (G6PDH) in regulating the levels of reduced form of glutathione (GSH) to the tolerance of calli from two reed ecotypes, Phragmites communis Trin. dune reed (DR) and swamp reed (SR), in a long-term salt stress. G6PDH activity was higher in SR callus than that of DR callus under 50–150 mM NaCl treatments. In contrast, at higher NaCl concentrations (300–600 mM), G6PDH activity was lower in SR callus. A similar profile was observed in GSH contents, glutathione reductase (GR) and glutathione peroxidase (GPX) activities in both salt-stressed calli. After G6PDH activity and expression were reduced in glycerol treatments, GSH contents and GR and GPX activity decreased strongly in both calli. Simultaneously, NaCl-induced hydrogen peroxide (H₂O₂) accumulation was also abolished. Exogenous application of H₂O₂ increased G6PDH, GR, and GPX activities and GSH contents in the control conditions and glycerol treatment. Diphenylene iodonium (DPI), a plasma membrane (PM) NADPH oxidase inhibitor, which counteracted NaCl-induced H₂O₂ accumulation, decreased these enzymes activities and GSH contents. Furthermore, exogenous application of H₂O₂ abolished the N-acetyl-l-cysteine (NAC)-induced decrease in G6PDH activity, and DPI suppressed the effect of buthionine sulfoximine (BSO) on induction of G6PDH activity. Western-blot analyses showed that G6PDH expression was stimulated by NaCl and H₂O₂, and blocked by DPI in DR callus. Taken together, G6PDH activity involved in GSH maintenance and H₂O₂ accumulation under salt stress. And H₂O₂ regulated G6PDH, GR, and GPX activities to maintain GSH levels. In the process, G6PDH plays a central role.     

Purification and characterization of the flavoenzyme glutathione reductase from rat liver.

Glutathione reductase from rat liver has been purified greater than 5000-fold in a yield of 20%. The molecular weights of the enzyme and its subunits were estimated to be 125,000 and 60,000, respectively, indicating that the native enzyme is a dimer. The enzyme molecular contains 2 FAD molecules, which are reducible by NADPH, GSH or dithioerythritol. The reduced flavin is instantaneously reoxidized by addition of GSSG. The steady state kinetic data are consistent with a branching reaction mechanism previously proposed for glutathione reductase from yeast (MANNERVIK, B. (1973) Biochem. Biophy. Res. Commun. 53, 1151-1158). This mechanism is also favored by the nonlinear inhibition pattern produced by NADP-+. However, at low GSSG concentrations the rate equation can be approximated by that of a simple ping pong mechanism. NADPH and the mixed disulfide of coenzyme A and GSH were about 10% as active as NADPH and GSSG, respectively, whereas some sulfenyl derivatives related to GSSG were less active as substrates. The pH activity profiles of these substrates differed from that of the NADPH-GSSG substrate pair.     

Glucose-6-phosphate dehydrogenase activity and NADPH/NADP+ ratio in liver and pancreas are dependent on the severity of hyperglycemia in rat

Hyperglycemia is associated with metabolic disturbances affecting cell redox potential, particularly the NADPH/NADP+ ratio and reduced glutathione levels. Under oxidative stress, the NADPH supply for reduced glutathione regeneration is dependent on glucose-6-phosphate dehydrogenase. We assessed the effect of different hyperglycemic conditions on enzymatic activities involved in glutathione regeneration (glucose-6-phosphate dehydrogenase and glutathione reductase), NADP(H) and reduced glutathione concentrations in order to analyze the relative role of these enzymes in the control of glutathione restoration. Male Sprague-Dawley rats with mild, moderate and severe hyperglycemia were obtained using different regimens of streptozotocin and nicotinamide. Fifteen days after treatment, rats were killed and enzymatic activities, NADP(H) and reduced glutathione were measured in liver and pancreas. Severe hyperglycemia was associated with decreased body weight, plasma insulin, glucose-6-phosphate dehydrogenase activity, NADPH/NADP+ ratio and glutathione levels in the liver and pancreas, and enhanced NADP+ and glutathione reductase activity in the liver. Moderate hyperglycemia caused similar changes, although body weight and liver NADP+ concentration were not affected and pancreatic glutathione reductase activity decreased. Mild hyperglycemia was associated with a reduction in pancreatic glucose-6-phosphate dehydrogenase activity. Glucose-6-phosphate dehydrogenase, NADPH/NADP+ ratio and glutathione level, vary inversely in relation to blood glucose concentrations, whereas liver glutathione reductase was enhanced during severe hyperglycemia. We conclude that glucose-6-phosphate dehydrogenase and NADPH/NADP+ were highly sensitive to low levels of hyperglycemia. NADPH/NADP+ is regulated by glucose-6-phosphate dehydrogenase in the liver and pancreas, whereas levels of reduced glutathione are mainly dependent on the NADPH supply.     

Antioxidative enzyme and glutathione S-transferase activities in diabetic rats exposed to long-term ASA treatment

Low-dose acetylsalicylic acid (ASA) treatment is a standard therapeutic approach in diabetes mellitus for prevention of long-term vascular complications. The aim of the present work was to investigate the effect of long-term ASA administration in experimental diabetes on activities of some liver enzymes: glutathione peroxidase (GSHPx), catalase, glucose-6-phosphate dehydrogenase (G6PDH) and glutathione S-transferase (GST). Blood glucose, glycated hemoglobin, as well as plasma ALT and AST activities increased in rats with streptozotocin-induced experimental diabetes. The long-term hyperglycemia resulted in decreased activities of GSHPx (by 26%), catalase (by 34%), GST (by 38%) and G6PDH (by 27%) in diabetic animals. We did not observe increased accumulation of membrane lipid peroxidation products or altered levels of reduced glutathione in livers. The linear correlation between blood glucose and glycated hemoglobin in diabetic animals was distorted upon ASA treatment, which was likely due to a chemical competition between nonenzymatic protein glycosylation and protein acetylation. The long-term ASA administration partially reversed the decrease in GSHPx activity, but did not influence the activities of catalase and GST in diabetic rats. Otherwise, some decrease in these parameters was noted in ASA-treated nondiabetic animals. Increased ASA-induced G6PDH activity was recorded in both diabetic and nondiabetic rats. While both glycation due to diabetic hyperglycemia and ASA-mediated acetylation had very similar effects on the activities of all studied enzymes but G6PDH, we conclude that non-enzymatic modification by either glucose or ASA may be a common mechanism of the observed convergence.     

Hypoglycaemic, antioxidative and nephroprotective effects of taurine in alloxan diabetic rabbits

The therapeutic potential of taurine was investigated under diabetic conditions. Alloxan diabetic rabbits were treated daily for three weeks with 1% taurine in drinking water. The following parameters were measured: 1) serum glucose, urea, creatinine and hydroxyl free radical (HFR) levels; 2) blood glutathione redox state; 3) urine albumin concentration; 4) hepatic and renal HFR levels, GSH/GSSG ratios and the activities of catalase, superoxide dismutase and the enzymes of glutathione metabolism; 5) renal NADPH oxidase activity; 6) the rates of renal and hepatic gluconeogenesis. Histological studies of kidneys were also performed. Taurine administration to diabetic rabbits resulted in 30% decrease in serum glucose level and the normalisation of diabetes-elevated rate of renal gluconeogenesis. It also decreased serum urea and creatinine concentrations, attenuated diabetes-evoked decline in GSH/GSSG ratio and abolished hydroxyl free radicals accumulation in serum, liver and kidney cortex. Animals treated with taurine exhibited elevated activities of hepatic gamma-glutamylcysteine syntetase and renal glutathione reductase and catalase. Moreover, taurine treatment evoked the normalisation of diabetes-stimulated activity of renal NADPH oxidase and attenuated both albuminuria and glomerulopathy characteristic of diabetes. In view of these data, it is concluded that: 1) diminished rate of renal gluconeogenesis seems to contribute to hypoglycaemic effect of taurine; 2) taurine-induced increase in the activities of catalase and the enzymes of glutathione metabolism is of importance for antioxidative action of this amino acid and 3) taurine nephroprotective properties might result from diminished renal NADPH oxidase activity. Thus, taurine seems to be beneficial for the therapy of both diabetes and diabetic nephropathy.     

Liver and colon pro- and anti-oxidant enzyme activities in rats after long-term ethylnitrosourea exposure

Liver and colon pro- and anti-oxidant enzyme activities were investigated in rats treated with ethylnitrosourea (ENU) (i.p.) (4 mg/kg body wt) for 6 months. The pro-oxidant enzymes (NADPH cytochrome c reductase, NADH cytochrome c reductase, NADH cytochrome b5 reductase and cytochrome P-4502E1and the antioxidant enzyme, superoxide dismutase (SOD) exhibited significantly increased activity in liver and colon. Glucose-6-phosphate dehydrogenase (G6PDH) and glutathione-S-transferase (GST) showed enhanced activity in liver, but decreased activity in colon. Glutathione peroxidase (GP) and glutathione reductase (GR) activities were significantly increased in colon, but decreased in liver. Catalase (CAT) activity while showed a significant increase in liver, exhibited only marginal increase in colon. Malondialdehyde (MDA) level was significantly elevated in both tissues.     

Changes in brown-adipose-tissue mitochondrial processes in streptozotocin-diabetes.

Yeast glucose-6-phosphate dehydrogenase was inhibited by low NADPH concentrations in cell-free extracts, and de-inhibited by GSSG; extensive dialysis of the crude extract did not diminish the GSSG effect. Immunoprecipitation of glutathione reductase abolished the de-inhibition of glucose-6-phosphate dehydrogenase by GSSG. Purified glucose-6-phosphate dehydrogenase was inhibited by NADPH but not de-inhibited by GSSG, and upon addition of pure glutathione reductase GSSG completely de-inhibited the glucose-6-phosphate dehydrogenase.     

Dehydroepiandrosterone improves hepatic antioxidant reserve and stimulates Akt signaling in young and old rats

This study examined, in the liver of young and old (3- and 24-month-old, respectively) healthy Wistar rats, the in vivo effect of dehydroepiandrosterone (DHEA) (10mg/kg body weight) administered subcutaneously for 5 weeks. Reduced (GSH) and oxidized (GSSG) glutathione levels, glucose-6-phosphate dehydrogenase (G6PDH), glutathione-S-transferase (GST), glutathione peroxidase (GPx) and catalase (CAT) activities, hydrogen peroxide concentration, GST and p-Akt/Akt immunocontent ratio were assessed in hepatic tissue. DHEA treatment significantly increased total glutathione content (17%) and GSH (22%) in 3- and 24-month-old treated groups when compared to control groups. The aging factor increased G6PDH (51%) and GPx (22%) activities as well as the hydrogen peroxide concentration (33%), independently of treatment. DHEA treatment increased p-Akt (54%) and p-Akt/Akt ratio (36%) immunocontents in both treated groups. Increased serum levels of alanine aminotransferase (ALT) in aged rats were reduced by DHEA treatment (34%).