A ferroptosis defense mechanism mediated by glycerol-3-phosphate dehydrogenase 2 in mitochondria.

Mechanisms of defense against ferroptosis (an iron-dependent form of cell death induced by lipid peroxidation) in cellular organelles remain poorly understood, hindering our ability to target ferroptosis in disease treatment. In this study, metabolomic analyses revealed that treatment of cancer cells with glutathione peroxidase 4 (GPX4) inhibitors results in intracellular glycerol-3-phosphate (G3P) depletion. We further showed that supplementation of cancer cells with G3P attenuates ferroptosis induced by GPX4 inhibitors in a G3P dehydrogenase 2 (GPD2)-dependent manner; GPD2 deletion sensitizes cancer cells to GPX4 inhibition-induced mitochondrial lipid peroxidation and ferroptosis, and combined deletion of GPX4 and GPD2 synergistically suppresses tumor growth by inducing ferroptosis in vivo. Mechanistically, inner mitochondrial membrane-localized GPD2 couples G3P oxidation with ubiquinone reduction to ubiquinol, which acts as a radical-trapping antioxidant to suppress ferroptosis in mitochondria. Taken together, these results reveal that GPD2 participates in ferroptosis defense in mitochondria by generating ubiquinol.

The 2',4'-dihydroxychalcone could be explored to develop new inhibitors against the glycerol-3-phosphate dehydrogenase from Leishmania species.

The enzyme glycerol-3-phosphate dehydrogenase (G3PDH) from Leishmania species is considered as an attractive target to design new antileishmanial drugs and a previous in silico study reported on the importance of chalcones to achieve its inhibition. Here, we report the identification of a synthetic chalcone in our in vitro assays with promastigote cells from Leishmania amazonensis, its biological activity in animal models, and docking followed by molecular dynamics simulation to investigate the molecular interactions and structural patterns that are crucial to achieve the inhibition complex between this compound and G3PDH. A molecular fragment of this natural product derivative can provide new inhibitors with increased potency and selectivity.

BAY 41-2272 activates host defence against local and disseminated Candida albicans infections

In our previous study, we have found that 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]-pyrimidin-4-ylamine (BAY 41-2272), a guanylate cyclase agonist, activates human monocytes and the THP-1 cell line to produce the superoxide anion, increasing in vitro microbicidal activity, suggesting that this drug can be used to modulate immune functioning in primary immunodeficiency patients. In the present work, we investigated the potential of the in vivo administration of BAY 41-2272 for the treatment of Candida albicans and Staphylococcus aureus infections introduced via intraperitoneal and subcutaneous inoculation. We found that intraperitoneal treatment with BAY 41-2272 markedly increased macrophage-dependent cell influx to the peritoneum in addition to macrophage functions, such as spreading, zymosan particle phagocytosis and nitric oxide and phorbol myristate acetate-stimulated hydrogen peroxide production. Treatment with BAY 41-2272 was highly effective in reducing the death rate due to intraperitoneal inoculation of C. albicans, but not S. aureus. However, we found that in vitro stimulation of peritoneal macrophages with BAY 41-2272 markedly increased microbicidal activities against both pathogens. Our results show that the prevention of death by the treatment of C. albicans-infected mice with BAY 41-2272 might occur primarily by the modulation of the host immune response through macrophage activation. .

Anti-obesity effects of hispidin and Alpinia zerumbet bioactives in 3T3-L1 adipocytes.

Obesity and its related disorders have become leading metabolic diseases. In the present study, we used 3T3-L1 adipocytes to investigate the anti-obesity activity of hispidin and two related compounds that were isolated from Alpinia zerumbet (alpinia) rhizomes. The results showed that hispidin, dihydro-5,6-dehydrokawain (DDK), and 5,6-dehydrokawain (DK) have promising anti-obesity properties. In particular, all three compounds significantly increased intracellular cyclic adenosine monophosphate (cAMP) concentrations by 81.2% ± 0.06%, 67.0% ± 1.62%, and 56.9% ± 0.19%, respectively. Hispidin also stimulated glycerol release by 276.4% ± 0.8% and inhibited lipid accumulation by 47.8% ± 0.16%. Hispidin and DDK decreased intracellular triglyceride content by 79.5% ± 1.37% and 70.2% ± 1.4%, respectively, and all three compounds inhibited glycerol-3-phosphate dehydrogenase (GPDH) and pancreatic lipase, with hispidin and DDK being the most potent inhibitors. Finally, none of the three compounds reduced 3T3-L1 adipocyte viability. These results highlight the potential for developing hispidin and its derivatives as anti-obesity compounds.

Inhibition of mitochondrial glycerol-3-phosphate dehydrogenase by α-tocopheryl succinate.

α-Tocopheryl succinate (TOS), a redox-silent analogue of vitamin E, suppresses cell growth in a number of clinical and experimental cancers, inhibits mitochondrial succinate dehydrogenase (SDH) and activates reactive oxygen species (ROS) generation. The aim of this study was to test whether TOS also inhibits glycerol-3-phosphate dehydrogenase (mGPDH), another flavoprotein-dependent enzyme of the mitochondrial respiratory chain because there are differences between electron transfer pathway from SDH and mGPDH to coenzyme Q pool. For our experiments brown adipose tissue mitochondria with high expression of mGPDH were used. Our data showed that inhibition of glycerol-3-phosphate (GP)-dependent oxygen consumption by TOS was more pronounced than the succinate (SUC)-dependent one (50% inhibition was reached at 10 µmol/l TOS vs. 80 µmol/l TOS, respectively). A comparison of the inhibitory effect of TOS on GP-oxidase, GP-cytochrome c oxidoreductase and GP-dehydrogenase activities showed that TOS directly interacts with the dehydrogenase. After TOS application the GP-dependent generation of ROS was highly depressed. It may thus be concluded that TOS-induced inhibition of mGPDH is more pronounced than TOS-induced inhibition of SDH and that the inhibitory effect of TOS for both substrates is exerted at different locations of the particular dehydrogenases. Our data indicate that the inhibition of mGPDH activity could also play a role in TOS-induced growth suppression in neoplastic cells.

Kefir inhibits 3T3-L1 adipocyte differentiation through down-regulation of adipogenic transcription factor expression.

BACKGROUND: Kefir, a traditional fermented milk composed of microbial symbionts, is reported to have various health benefits such as anti-tumour, anti-inflammatory, anti-neoplastic and pro-digestive effects. In this study, to elucidate the effects of kefir on adipocyte differentiation and lipid accumulation, three fractions were prepared from kefir culture broth. The inhibitory effects of kefir liquid culture broth fraction (Fr-1), soluble fraction (Fr-2) and insoluble fraction (Fr-3), prepared by sonication of kefir solid culture broth, on adipocyte differentiation in 3T3-L1 preadipocytes were examined. RESULTS: Fr-3 (0.1 mg mL(-1)) significantly decreased lipid accumulation and glycerol-3-phosphate dehydrogenase (GPDH) activity by 60 and 68% respectively without affecting cell viability. In addition, Fr-3 treatment down-regulated the mRNA expression of adipogenic transcription factors including C/EBPα (32%), PPARγ (46%) and SREBP-1c (34%) during adipocyte differentiation compared with untreated control cells. The mRNA expression of adipocyte-specific genes (aP2, FAS and ACC) was also clearly decreased. CONCLUSION: The results suggest that the insoluble fraction of kefir (Fr-3) mediates anti-adipogenic effects through the inhibition of adipocyte differentiation, partly via suppression of the C/EBPα-, SREBP-1c- and PPARγ-dependent pathways.

Caffeic acid phenethyl ester suppresses the production of adipocytokines, leptin, tumor necrosis factor -alpha and resistin, during differentiation to adipocytes in 3T3-L1 cells.

We previously reported that caffeic acid phenethyl ester (CAPE) suppresses 3T3-L1 differentiation to adipocytes through the inhibition of peroxisome proliferator-activated receptor (PPAR) gamma, CCAAT/enhancer-binding protein (C/EBP) alpha, fatty acid synthase (Fas) and adipocytes-specific fatty acid binding protein 2 (aP2) expressions (Juman et al., Biol. Pharm. Bull., 33, 1484-1488 (2010)). In the present study, we confirmed that CAPE had inhibitory effects on increased glycerol-3-phosphate dehydrogenase (GPDH) activity and an increased insulin receptor substrate 1 (IRS-1). Our data show that treatment with 50 µM CAPE significantly reduced the levels of leptin (p<0.05), resistin (p<0.05) and tumor necrosis factor (TNF)-alpha (p<0.05) which are known to aid adipocytokines production in adipocytes. In 3T3-L1 cells, treatment of CAPE decreased the triglyceride deposition similar to resveratrol, which is known to have an inhibitory effect on 3T3-L1 differentiation to adipocytes. In conclusion, we found that CAPE suppresses the production and secretion of adipocytokines from mature adipocytes in 3T3-L1 cells.

Salacia reticulata and its polyphenolic constituents with lipase inhibitory and lipolytic activities have mild antiobesity effects in rats.

Salacia (S.) reticulata, a Hippocrateaceae plant distributed in Sri Lankan and Indian forests, has been used as a supplementary food in Japan to prevent obesity and diabetes. We examined the antiobesity effects of the hot water-soluble extract (SRHW) from the roots of S. reticulata using obese rat models and an in vitro study. Body weight (P = 0.07) and periuterine fat storage (P = 0.10) in female Zucker fatty rats (8-9 wk old) tended to be suppressed by oral administration of SRHW (125 mg/kg) for 27 d. Male rats fed a high fat diet were not affected by SRHW. Furthermore, SRHW inhibited porcine pancreatic lipase (PL), rat adipose tissue-derived lipoprotein lipase (LPL) and glycerophosphate dehydrogenase (GPDH) activities with 50% inhibitory concentrations (IC(50)) of 264 (95% confidence limits: 203-393) mg/L, 15 (12-18) mg/L and 54 (35-85) mg/L, respectively, but did not inhibit hormone-sensitive lipase activity in rat adipose tissue. Next, we examined the effects of polyphenols, di- and triterpenes and salacinol isolated from the roots of S. reticulata on lipid metabolizing enzymes and lipolysis. (-)-Epigallocatechin and (-)-epicatechin-(4beta-->8)-(-)-4'-O-methylepigallocatechin inhibited PL activity with IC(50) of 88 (not calculated) and 68 (26-122) mg/L, respectively. (-)-Epicatechin, 3beta, 22beta-dihydroxyolean-12-en-29-oic acid and the tannin fraction inhibited LPL activity with IC(50) of 81 (54-214), 89 (62-214) and 35 (24-62) mg/L. Only the tannin fraction inhibited GPDH activity with an IC(50) of 6.8 (3.4-10.9) mg/L. These constituents may be involved in the lipase and GPDH inhibitory activities of SRHW. On the other hand, SRHW at 100 mg/L tended to enhance lipolysis in rat adipocytes (P = 0.06). Significant lipolytic effects were exerted by mangiferin, (-)-4'-O-methylepigallocatechin and maytenfolic acid at 100 mg/L (P < 0.01). In conclusion, polyphenolic compounds may be involved in the antiobesity effects of SRHW in rats through inhibition of fat metabolizing enzymes (PL, LPL and GPDH) and enhanced lipolysis.

Redox regulation of cytosolic glycerol-3-phosphate dehydrogenase: Cys(102) is the target of the redox control and essential for the catalytic activity.

Cytosolic glycerol-3-phosphate dehydrogenase (cG3PDH) occupies the branch point between the glycolytic pathway and triglyceride biosynthesis. However, the regulatory mechanism of the cG3PDH activity has remained obscure. Here we report that cG3PDH is efficiently inhibited by modification of the thiol group through a redox mechanism. In this study, we found that sodium selenite and nitric oxide (NO) donors such as S-nitroso-N-acetylpenicillamine and 3-morpholinosydnonimine inhibited cG3PDH activity, and that similar effects could be achieved with selenium metabolites such as selenocysteine and selenomethionine. Furthermore, we found that reducing agents, such as dithiothreitol and beta-mercaptoethanol, restored the cG3PDH activity suppressed by selenite and NO both in vitro and in cultured cells. Buthionine sulfoximine depleted levels of both reduced glutathione and the oxidized form but had no effect on the suppression of cG3PDH activity by selenite in cultured cells. Moreover, thiol-reactive agents, such as N-ethylmaleimide and o-iodosobenzoic acid, blocked the enzyme activity of cG3PDH through the modification of redox-sensitive cysteine residues in cG3PDH. The inhibitor of NO synthase, L-N(G)-nitro-arginine, restored the cG3PDH activity inhibited by NO in cultured cells, whereas the inhibitor of guanylyl cyclase, 1H-[1,2,4] oxadiazole[4,3-alpha] quinoxalin-1-one (ODQ), has no effect. NO directly inhibits cG3PDH activity not via a cGMP-dependent mechanism. Finally, using site-directed mutagenesis, we found that Cys(102) of cG3PDH was sensitive to both selenite and NO. From the results, we suggest that cG3PDH is a target of cellular redox regulation.

Affinity chromatography using trypanocidal arsenical drugs identifies a specific interaction between glycerol-3-phosphate dehydrogenase from Trypanosoma brucei and Cymelarsan.

A 36-kDa protein was isolated by affinity chromatography using Cymelarsan, an arsenical drug currently used in African trypanosomiasis treatment, as ligand. This protein was identified as glycerol-3-phosphate dehydrogenase. Trypanosomal glycerol-3-phosphate was bound covalently, whereas its counterpart from rabbit muscle bound by ionic interaction. Arsenical drugs inhibit the enzyme in a dose-dependent manner. Oxidation of cysteine residues protects against inactivation without significantly diminishing enzymic activity. Drug concentrations giving 50% inhibition of the dehydrogenase activity were determined for the enzyme from both Trypanosoma brucei and rabbit and indicate a higher sensitivity of the trypanosomal enzyme to arsenical drugs and thiol reagents. MS was used to identify residues of glycerol-3-phosphate dehydrogenase bound by Cymelarsan; they are not conserved in the mammalian enzyme.

Effect of NADH-X on cytosolic glycerol-3-phosphate dehydrogenase.

At pH 7.05 NADH-X prepared by incubating NADH with glyceraldehyde-3-phosphate dehydrogenase (E.C. 1.2.1.12) was a potent noncompetitive inhibitor, with respect to coenzyme, of NADPH oxidation by pure rabbit muscle cytosolic glycerol-3-phosphate dehydrogenase (E.C. 1.1.1.8) and also a potent inhibitor of NADPH oxidation catalyzed by this enzyme in a rat pancreatic islet cytosolic fraction. It was a much less potent inhibitor of NADPH oxidation catalyzed by this enzyme in a rat liver cytosolic fraction and of NADH oxidation catalyzed by this enzyme from all three sources. Glycerol-3-phosphate dehydrogenase purified from muscle cytosol contains tightly bound NADH-X, NAD, and ADP-ribose, each in amounts of about 0.1 mol per mole of enzyme polypeptide chain. A deproteinized supernatant of this enzyme contained these three ligands and produced the same type of inhibition of the enzyme described above for prepared NADH-X with a Ki, in the reaction with NADPH at pH 7.05, in the range of 0.2 microM with respect to the total concentration of ligands ([ADP-ribose] + [NAD] + [NADH-X] = 0. 2 microM). However, only the NADH-X component could account for the potent inhibition because NAD, ADP-ribose, and the primary acid product (which can be produced from NADH-X) each had a Ki considerably higher than 0.2 microM. Although at pH 7.05 NADH-X inhibited NADPH oxidation considerably more than NADH oxidation, the reverse was the case at pH 7.38. Since the enzyme purified from muscle contains tightly bound NADH-X, NADH-X might become attached to the enzyme in vivo where it could play a role in regulating the ratio of NADH to NADPH oxidation of the enzyme.

Inhibition of mitochondrial cytochrome C oxidase by dicarbanonaborates.

Dicarbanonaborates inhibit the mitochondrial cytochrome c oxidase activity. In contrast to mitochondrial ATPase or glycerol phosphate dehydrogenase, inhibition of cytochrome c oxidase was not competitive and the residual, drug-insensitive activity was higher. These results indicate that dicarbanonaborates inhibit various mitochondrial membrane-bound enzymes through different mechanisms.

Selective interaction of glycosomal enzymes from Trypanosoma brucei with hydrophobic cyclic hexapeptides.

Hydrophobic cyclic hexapeptides have been reported to selectively inhibit glycosomal triosephosphate isomerase from Trypanosoma brucei (Kuntz et al, 1992, Eur. J. Biochem., 207, 441-447). Here it is shown that this inhibition is not due to a specific interaction between the enzyme and soluble hydrophobic cyclic hexapeptides, but that it is the result of a coprecipitation of trypanosome triosephosphate isomerase with cyclic hexapeptides when the solubilities of the latter are exceeded. A study of the interaction of these hexapeptides with other glycosomal enzymes revealed that several of them, such as phosphoglycerate kinase and hexokinase, also coprecipitated with these peptides, whereas most of the homologous enzymes from other organisms did not coprecipitate, nor were they inactivated.

Stimulation of oxidative metabolism by thyroid hormones in an apodan amphibian, Gegenophis carnosus (Beddome).

Administration of different doses of L-thyroxine (T4) and triiodo-L-thyronine (T3) in vivo in G. carnosus stimulated the activities of cytochrome oxidase, alpha-glycerophosphate dehydrogenase (alpha-GPDH), succinate dehydrogenase (SDH), and Mg2+ adenosine triphosphatase (Mg2+ ATPase) and inhibited the activity of malate dehydrogenase (MDH). While a low dose of thiouracil administration produced a stimulatory effect on cytochrome oxidase and alpha-GPDH activities, a higher dose of thiouracil significantly inhibited the activities of cytochrome oxidase, alpha-GPDH, SDH, Mg2+ ATPase, and MDH. Injection of T4 or T3 into thiouracil-treated animals significantly restored the stimulatory effect of thyroid hormones on oxidative enzyme activities. It is suggested that thyroid hormones in vivo increase and that thiouracil decreases the oxidative capacity of hepatic mitochondria of G. carnosus.

Second stage tumor promoters: differences in biological potency and phorbol ester receptor affinity in C6 cells.

We have shown that the second stage tumor promoters mezerein (MEZ) and phorbol 12-retinoate 13-acetate (PRA) inhibit the gluccocorticoid-induced increase in glycerol phosphate dehydrogenase (GPDH) activity in C6 rat glioma cells with ED 50-values of 3.9 and 2.9 nM, respectively. Phorbol 12-myristate 13-acetate (PMA) was 10-fold less potent. MEZ was likewise more potent than PMA for inhibition of cAMP formation in response to isoproterenol. Binding competition studies using [3H]phorbol 12,13-dibutyrate ([3H]PDBu) yielded apparent Ki-values for MEZ and PRA of 50-70 nM. The large difference between the biological potencies of MEZ and PRA and their affinity for the major phorbol ester receptor suggest they may be acting through a more complicated mechanism in these cells.

Activation of mitochondrial glycerol 3-phosphate dehydrogenase by cadmium ions.

Mitochondrial glycerol 3-phosphate dehydrogenase (EC 1.1.2.1.) requires Ca2+ ions for its activity. Cadmium ions also have activatory effect on the enzyme. They activate the glycerol 3-phosphate dehydrogenase in a very narrow concentration range (1-2 mmol/l). As contrasted with calcium, strong inhibitory effect occurred at higher concentrations (3-4 mmol/l). The inhibition induced by cadmium ions was completely reversible by washing of the mitochondria.

Increased mitochondrial glycerol phosphate dehydrogenase activity in insulinomas of two hypoglycemic infants.

The activities of flavin-linked, mitochondrial glycerol phosphate dehydrogenase in islet cell adenomas from two infants were 8 and 16 times higher than those in their uninvolved pancreases. The enzyme in the insulinomas, but not that in the nearby pancreases, was inhibited by diazoxide, which is a known inhibitor of insulin secretion in vitro and in vivo. These results provide preliminary evidence that the enzyme may be used as an adjunct to histology for identifying human insulin-secreting tissues. Its sensitivity to diazoxide adds specificity to the test. Taken together with previous work with animal pancreatic islets, the results of the present study suggest that the glycerol phosphate shuttle plays an important role in stimulus-secretion coupling in the pancreatic beta-cell.

Partial purification, substrate specificity and regulation of alpha-L-glycerolphosphate dehydrogenase from Saccharomyces carlsbergensis.

alpha-L-Glycerolphosphate dehydrogenase (sn-glycerol-3-phosphate:NAD+ 2-oxidoreductase, EC 1.1.1.8) from Saccharomyces carlsbergensis was purified 400-fold. The enzyme preparation is free of interfering activities, such as glyceraldehyde phosphate dehydrogenase, alcohol dehydrogenase, triose phosphate isomerase and glycerolphosphatase. At pH 7.0 it is specific for NADH (Km = 0.027 mM with 0.8 mM dihydroxyacetone phosphate) and dihydroxyacetone phosphate (Km = 0.2 mM with 0.2 mM NADH). Between pH 5.0 and 6.0 the enzyme functions with NADPH, but only at 7% of the rate with NADH. Various anions (I- greater than SO42- greater than Br- greater than Cl-) act as inhibitors competing with the substrate dihydroxyacetone phosphate. Inorganic phosphate (Ki = 0.1 mM), pyrophosphate and arsenate are strong inhibitors. The nucleotides ATP and ADP are also inhibitory, but their action seems to be of the same type as the general anion competition (Ki = 0.73 mM for ATP). The results are consistent with the notion that the enzyme may regulate the redox potential of the NAD+/NADH couple during fermentation.

The role of cysteine residues in the catalytic activity of glycerol-3-phosphate dehydrogenase.

Glycerol-3-phosphate dehydrogenase (sn-glycerol-3-phosphate:NAD+ 2-oxido-reductase, EC 1.1.1.8) has been shown to be sensitive to inhibition by iodoacetate. The reaction of the enzyme with iodoacetate, which appears to be a simple bimolecular process, is accompanied by a corresponding loss of enzyme activity. In addition to changes in activity, the alkylation reaction was monitored by the incorporation of radioactivity from iodo[2-14C]acetate, by changes in amino acid composition, and by changes in the content of free sulfhydryl groups. It is concluded that there are two cysteine residues in the native dimeric enzyme which are essential for enzymic activity. The rate of inactivation was relatively insensitive to the presence of various compounds with the exception of NADH which markedly inhibited the reaction. Kinetic and binding studies showed that the binding of NADH prevents alkylation and, conversely, alkylation prevents NADH binding. From the pH dependence of the alkylation reaction, the pKa of the essential sulfhydryl groups was calculated to be 8.5 and it is suggested that the binding of coenzyme is independent of the state of ionization of these groups.