Tricarboxylic acid cycle dehydrogenases inhibition by naringenin: experimental and molecular modelling evidence.

The study of polyphenols' effects on health has been gaining attention lately. In addition to reacting with important enzymes, altering the cell metabolism, these substances can present either positive or negative metabolic alterations depending on their consumption levels. Naringenin, a citrus flavonoid, already presents diverse metabolic effects. The objective of this work was to evaluate the effect of maternal naringenin supplementation during pregnancy on the tricarboxylic acid cycle activity in offspring's cerebellum. Adult female Wistar rats were divided into two groups: (1) vehicle (1 ml/kg by oral administration (p.o.)) or (2) naringenin (50 mg/kg p.o.). The offspring were euthanised at 7th day of life, and the cerebellum was dissected to analyse citrate synthase, isocitrate dehydrogenase (IDH), α-ketoglutarate dehydrogenase (α-KGDH) and malate dehydrogenase (MDH) activities. Molecular docking used SwissDock web server and FORECASTER Suite, and the proposed binding pose image was created on UCSF Chimera. Data were analysed by Student's t test. Naringenin supplementation during pregnancy significantly inhibited IDH, α-KGDH and MDH activities in offspring's cerebellum. A similar reduction was observed in vitro, using purified α-KGDH and MDH, subjected to pre-incubation with naringenin. Docking simulations demonstrated that naringenin possibly interacts with dehydrogenases in the substrate and cofactor binding sites, inhibiting their function. Naringenin administration during pregnancy may affect cerebellar development and must be evaluated with caution by pregnant women and their physicians.

α-Viniferin and resveratrol induced alteration in the activities of some energy metabolism related enzymes in the cestode parasite Raillietina echinobothrida.

α-Viniferin (AVF) and its monomer resveratrol (RESV) are natural phytostilbenes produced by several plants in response to injury or under the influence of pathogens such as bacteria or fungi. Our earlier studies have revealed that both the compounds exert anthelmintic activity through alterations of cestode tegument and its associated enzymes. The present study investigates the effects of these phytochemicals on some energy metabolism related enzymes in the fowl tapeworm, Raillietina echinobothrida. The phytostilbenes AVF, RESV and the reference drug praziquantel (PZQ) were tested against some selected enzymes i.e., phosphoenolpyruvate carboxykinase (PEPCK), lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) of R. echinobothrida. Exposure of the tapeworm to AVF, RESV and PZQ causes reduction in activity of PEPCK to the extent of 40.57/41.96, 24.58/23.75 and 41.11/13.47%, respectively, and LDH up to 48.95/16.25, 38.31/38.42 and 45.67/41.87%, respectively, at the time of paralysis. Whereas activity of MDH decreased by 34.22/37.7, 39.1/35.24 and 28.83/19.26%, respectively. Decrease in activities of LDH and MDH was also visible through histochemical observations. The results suggest that both the phytochemicals interfere with the energy transducing pathways by inhibiting the studied energy metabolism related enzymes of the parasite.

ortho-Carboranylphenoxyacetanilides as inhibitors of hypoxia-inducible factor (HIF)-1 transcriptional activity and heat shock protein (HSP) 60 chaperon activity.

ortho-Carboranylphenoxy derivatives were synthesized and evaluated for their ability to inhibit hypoxia-induced HIF-1 transcriptional activity using a cell-based reporter gene assay. Among the compounds synthesized, compound 1d showed the most significant inhibition of hypoxia-induced HIF-1 transcriptional activity with the IC50 of 0.53µM. Furthermore, compound 1h was found to possess the most significant inhibition of heat shock protein (HSP) 60 chaperon activity among the reported inhibitors: the IC50 toward the porcine heart malate dehydrogenase (MDH) refolding assay was 0.35µM.

The characterization of neuroenergetic effects of chronic L-tyrosine administration in young rats: evidence for striatal susceptibility.

Tyrosinemia type II is an inborn error of metabolism caused by a deficiency in hepatic cytosolic aminotransferase. Affected patients usually present a variable degree of mental retardation, which may be related to the level of plasma tyrosine. In the present study we evaluated effect of chronic administration of L-tyrosine on the activities of citrate synthase, malate dehydrogenase, succinate dehydrogenase and complexes I, II, II-III and IV in cerebral cortex, hippocampus and striatum of rats in development. Chronic administration consisted of L-tyrosine (500 mg/kg) or saline injections 12 h apart for 24 days in Wistar rats (7 days old); rats were killed 12 h after last injection. Our results demonstrated that L-tyrosine inhibited the activity of citrate synthase in the hippocampus and striatum, malate dehydrogenase activity was increased in striatum and succinate dehydrogenase, complexes I and II-III activities were inhibited in striatum. However, complex IV activity was increased in hippocampus and inhibited in striatum. By these findings, we suggest that repeated administrations of L-tyrosine cause alterations in energy metabolism, which may be similar to the acute administration in brain of infant rats. Taking together the present findings and evidence from the literature, we hypothesize that energy metabolism impairment could be considered an important pathophysiological mechanism underlying the brain damage observed in patients with tyrosinemia type II.

Human mitochondrial NAD(P)(+)-dependent malic enzyme participates in cutaneous melanoma progression and invasion.

Cutaneous melanoma is the most life-threatening neoplasm of the skin, accounting for most of the skin cancer deaths. Accumulating evidence suggests that targeting metabolism is an appealing strategy for melanoma therapy. Mitochondrial NAD(P)(+)-dependent malic enzyme (ME2), an oxidative decarboxylase, was evaluated for its biological significance in cutaneous melanoma progression. ME2 mRNA and protein expression significantly increased during melanoma progression, as evidenced by Gene Expression Omnibus analysis and immunohistochemistry on clinically annotated tissue microarrays, respectively. In addition, ME2 knockdown attenuated melanoma cell proliferation in vitro. ME2 ablation resulted in reduced cellular ATP levels and elevated cellular reactive oxygen species production, which activated the AMP-activated protein kinase pathway and inhibited acetyl-CoA carboxylase. Furthermore, ME2 expression was associated with cell migration and invasion. ME2 knockdown decreased anchorage-independent growth in vitro and tumor cell growth in vivo. These results suggested that ME2 might be an important factor in melanoma progression and a novel biomarker of invasion.

Fluvoxamine alters the activity of energy metabolism enzymes in the brain

Objective: Several studies support the hypothesis that metabolism impairment is involved in the pathophysiology of depression and that some antidepressants act by modulating brain energy metabolism. Thus, we evaluated the activity of Krebs cycle enzymes, the mitochondrial respiratory chain, and creatine kinase in the brain of rats subjected to prolonged administration of fluvoxamine. Methods: Wistar rats received daily administration of fluvoxamine in saline (10, 30, and 60 mg/kg) for 14 days. Twelve hours after the last administration, rats were killed by decapitation and the prefrontal cortex, cerebral cortex, hippocampus, striatum, and cerebellum were rapidly isolated. Results: The activities of citrate synthase, malate dehydrogenase, and complexes I, II-III, and IV were decreased after prolonged administration of fluvoxamine in rats. However, the activities of complex II, succinate dehydrogenase, and creatine kinase were increased. Conclusions: Alterations in activity of energy metabolism enzymes were observed in most brain areas analyzed. Thus, we suggest that the decrease in citrate synthase, malate dehydrogenase, and complexes I, II-III, and IV can be related to adverse effects of pharmacotherapy, but long-term molecular adaptations cannot be ruled out. In addition, we demonstrated that these changes varied according to brain structure or biochemical analysis and were not dose-dependent. .

Effect of five cysteine-containing compounds on three lipogenic enzymes in Balb/cA mice consuming a high saturated fat diet.

The in vivo effects of N-acetyl cysteine (NAC), S-allyl cysteine, S-ethyl cysteine (SEC), S-methyl cysteine (SMC), and S-propyl cysteine (SPC) against hyperlipidemia development and oxidation stress in Balb/cA mice consuming a high saturated fat diet were examined. The influence of these agents on plasma levels of glucose, insulin, uric acid, TG, cholesterol, and the activity of three lipogenic enzymes--glucose-6-phosphate dehydrogenase, malic enzyme, and FA synthase--was determined. All mice consumed the coconut oil-basd, high saturated fat diet, water, and cysteine or one of the five cysteine-containing compounds for 4 wk. The diet with 18% saturated fat significantly elevated the activity of three lipogenic enzymes and significantly increased TG and cholesterol biosynthesis in plasma and liver (P < 0.05). When compared with the water and cysteine groups, the treatments from five cysteine-containing agents significantly reduced high saturated fat diet-increased malic enzyme and FA synthase activities, and significantly lowered TG levels in plasma and liver (P< 0.05); however, only NAC, SAC, and SMC treatments significantly reduced cholesterol levels in plasma and liver (P < 0.05). The five cysteine-containing agents significantly restored high saturated fat diet-decreased glutathione peroxidase (GPX) activity in liver (P< 0.05); however, only SMC and SPC significantly restored GPX activity in heart and kidney (P< 0.05). These agents also significantly improved high saturated fat diet-related hyperglycemia, hyperuricemia, and oxidation stress (P < 0.05). These data support the hypothesis that these compounds are potential multiply-protective agents for hyperlipidemia prevention or therapy.

Diets rich in polyunsaturated fatty acids: effect on hepatic metabolism in rats.

OBJECTIVE: We investigated the effect of diets rich in omega-6 and omega-3 fatty acids on hepatic metabolism. METHODS: Male Wistar rats, just weaned, were fed ad libitum for 8 wk with one of the following diets: rat chow (C), rat chow containing 15% (w/w) soybean oil (S), rat chow containing 15% (w/w) fish oil (F), and rat chow containing 15% soy bean and fish oil (SF; 5:1, w/w). Casein was added to the fatty diets to achieve the same content of protein (20%) as the control chow. The rats were killed by decapitation, and the hepatic tissue was removed and weighed. Tissue lipid, glycogen, and protein content, in vivo lipogenesis rate, and adenosine triphosphate citrate lyase and malic enzyme activities were evaluated. Plasma total lipids, triacylglycerol, and cholesterol concentrations were assessed. RESULTS: Body weight gain was higher in F and SF than in C and S rats. Liver weight, lipid content, and lipogenesis rate increased in F and SF rats, although adenosine triphosphate citrate lyase activity decreased. Glycogen concentration decreased in S, F, and SF rats compared with C rats. Plasma total lipids and triacylglycerol concentrations were lower in F and SF than in C rats. Total and high-density lipoprotein cholesterol (HDL-C) plasma levels decreased in F rats, with maintenance of the total:HDL-C ratio. In SF rats, an increase in HDL-C led to a lower total:HDL-C ratio. CONCLUSIONS: These results indicated that an enrichment of the diet with omega-3 polyunsaturated fatty acids produces hypolipidemia but may cause changes in liver metabolism that favor lipid deposition. They also suggested that the addition of a small amount of eicosapentaenoic and docosahexaenoic polyunsaturated fatty acids to an omega-6-rich diet further improve the circulating lipid profile, in comparison with an omega-3-rich diet, but it does not prevent excess liver lipid accumulation.

Effect of ozone on metabolic activities of Escherichia coli K-12.

Escherichia coli K-12 cell suspensions in buffer were exposed to ozone at a concentration of 600 ppm. Measurements were made of cell viability, glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase, lactate dehydrogenase, glutathione disulfide reductase, nonprotein sulfhydryl and total sulfhydryl compounds. Cell viability was not affected when E. coli K-12 was exposed to ozone for less than 10 minutes. The most sensitive parameter was glyceraldehyde-3-phosphate dehydrogenase followed by nonprotein sulfhydryl and total sulfhydryl compounds. Effects on malate dehydrogenase, lactate dehydrogenase and glutathione disulfide reductase were negligible. Cell survival and induction of lipid oxidation were also determined using two strains of E. coli K-12 (rec A, deficient in DNA repair and wild-type). The extent of membrane lipid oxidation correlated with cell viability in a dose-dependent manner and the survival curves of both strains showed similar sensitivity to ozone. The data suggest that the sulfhydryl group in the membrane is the primary target of ozone attack. Rec A DNA repair system does not appear to play a role in ozone resistance.

Increased efficiency of GroE-assisted protein folding by manganese ions.

This study addresses the role of ATP-bound and free Mg2+ and Mn2+ ions in the activation and modulation of chaperonin-assisted refolding of urea-denatured malate dehydrogenase. As compared with Mg2+, Mn2+ ions caused a significant increase in the rate of GroE-assisted malate dehydrogenase refolding and, concomitantly, a decrease in the rate of ATP hydrolysis. Moreover, Mn2+ increases the affinity of GroES for GroEL, even in the presence of saturating amounts of Mg2+. Chemical cross-linking showed that lower concentrations of Mn-ATP as compared with Mg-ATP are needed to form both asymmetric GroEL14GroES7 and symmetric GroEL14(GroES7)2 particles. The manganese-dependent increase in the rate of protein folding concurred with a specific increase in the amount of symmetric GroEL14-(GroES7)2 particles detected in a chaperonin solution. Thus, Mn2+ is a cofactor that can markedly increase the efficiency of the chaperonin reaction in vitro. Mn2+ ions can serve as an important tool for analyzing the molecular mechanism and the structure of chaperonins.

Cysteines of chloroplast NADP-malate dehydrogenase form mixed disulfides.

Chloroplast NADP-malate dehydrogenase (NADP-MDH) from pea and from spinach was N-terminally truncated by limited proteolysis with Staphylococcus aureus protease V8. The resulting monomeric enzymes lacking, respectively, the 37 and 38 N-terminal amino acids were inactive. Reduction and addition of low concentrations of guanidine-HCl (50-100 mM) resulted in a highly active enzyme of 850 units per mg protein. Equilibration of the truncated enzyme with various glutathione (GSH) redox buffers and assaying its activity in the presence of guanidine-HCl was used to establish the existence of protein-GSH mixed disulfides. This finding was further confirmed using incorporation of radioactively labelled thiol. The possible function of such cysteine modifications under oxidative stress and their regeneration by the thioredoxin system in the light is discussed.

Refolding and recognition of mitochondrial malate dehydrogenase by Escherichia coli chaperonins cpn 60 (groEL) and cpn10 (groES).

In vitro refolding of pig mitochondrial malate dehydrogenase is investigated in the presence of Escherichia coli chaperonins cpn60 (groEL) and cpn10 (groES). When the enzyme is initially denatured with 3 M guanidinium chloride, chaperonin-assisted refolding is 100% efficient. C.d. spectroscopy reveals that malate dehydrogenase is almost unfolded in 3 M guanidinium chloride, suggesting that a state with little or no residual secondary structure is the optimal 'substrate' for chaperonin-assisted refolding. Malate dehydrogenase denatured to more highly structured states proves to refold less efficiently with chaperonin assistance. The enzyme is shown not to aggregate under the refolding conditions, so that losses in refolding efficiency result from irreversible misfolding. Evidence is advanced to suggest that the chaperonins are unable to rescue irreversibly misfolded malate dehydrogenase. A novel use is made of 100 K Centricon concentrators to study the binding of [14C]acetyl-labelled malate dehydrogenase to groEL by an ultrafiltration binding assay. Analysis of the data by Scatchard plot shows that acetyl-malate dehydrogenase, which has previously been extensively unfolded with guanidinium chloride, binds to groEL at a specific binding site(s). At saturation, one acetyl-malate dehydrogenase homodimer (two polypeptides) is shown to bind to each groEL homooligomer with a binding constant of approx. 10 nM.

The effect of changes in iron redox state on the activity of enzymes sensitive to modification of SH groups.

Iron ions in micromolar concentrations induced a rapid and selective inhibition of the activity of skeletal muscle creatine kinase (CK), sarcoplasmic reticulum (SR) Ca(2+)-ATPase, and pyruvate kinase (PK). This effect of iron was dependent on the presence of adenine nucleotides and on the redox state of iron. Changing the redox state of the media created different Fe2+/Fe3+ ratios which selectively depressed different enzymes: depression of PK activity occurred when iron was predominantly in its reduced form and, consequently, when there was a high Fe2+/Fe3+ ratio; depression of SR Ca2+ uptake and SR Ca(2+)-ATPase activity occurred when the Fe2+/Fe3+ ratio was close to 1; depression of CK activity occurred when iron was predominantly in its oxidized form and the Fe2+/Fe3+ ratio was low. All iron-sensitive enzymes possessed sulfhydryl groups, accessible to N-ethylmaleimide (NEM), which were essential for their activity. The rate of inhibition of enzyme activity with NEM increased in the order PK < Ca(2+)-ATPase < CK. Iron-induced depression of CK and PK activities was reversible by dithiotreithol. Results suggest that changes in the redox state of cellular microenvironments, which inevitably occur during reperfusion of ischemic tissue or rapid increase in tissue oxygen consumption, may selectively depress the activity of several enzymes bearing SH groups that are sensitive to modifications and that are essential for their activity. Iron-induced depression of enzyme activity depends on the availability of iron bound to adenine nucleotides and possibly to other low molecular weight chelators and on the Fe2+/Fe3+ ratio generated by the induced redox change.

The effects of a biological response modifier, OK-432, on tumor-induced alterations in the host metabolism.

The effects of multicytokine inducer, OK-432, on tumor-induced metabolic alterations were studied by assessing three key regulatory enzymes of gluconeogenesis, de novo fatty acid synthesis and the triglyceride clearance pathways. Two Klinish Einheit (KE) of OK-432 was subcutaneously injected on alternate days, for 10 days, into Fischer 344 rats with or without methylcholanthrene-induced sarcoma. At the time of sacrifice, the tumors accounted for approximately 23% of their total body weight. The injections of OK-432 did not affect the amount of food intake in either the tumor bearers or the controls. The tissue lipoprotein lipase activities in the epididymal fat pads of the tumor bearers were significantly decreased compared with the controls (P < 0.01). Phosphoenolpyruvate carboxykinase activity in the liver was significantly increased (P < 0.01), while malic enzyme activity tended to be decreased in the tumor bearers compared with the controls. However, there were no significant differences in those activities depending on the OK-432 injections, even though OK-432 induced tumor necrosis factor (TNF) and increased cytotoxic activities in the mesenteric lymph nodes as well as in the spleen. Thus, although the role of monokines in inducing cancer cachexia is not yet clearly understood, OK-432 was not able to revert the tumor-induced metabolic alterations which lead to tissue wasting and cancer cachexia.