Phenethyl isothiocyanate protects against H2O2-induced insulin resistance in 3T3-L1 adipocytes

Obesity is associated with systemic oxidative stress and leads to insulin resistance. Phenethyl isothiocyanate (PEITC), a natural dietary isothiocyanate, has been shown to have beneficial effects in improving cellular defense activities against oxidative stress through activation of nuclear factor erythroid-2 related factor 2 (Nrf2) pathway. However, little evidence exists if the antioxidative activity has beneficial effects on glucose metabolism. Here, we tested the preventive potential of PEITC for impaired insulin-induced glucose uptake by oxidative stress in 3T3-L1 adipocytes. Treatment with PEITC increased the expression of antioxidative enzymes regulated by Nrf2 such as γ-glutamylcysteine-synthetase, heme oxygenase 1, NAD(P)H:quinone oxidoreductase 1 and glutathione S-transferase, and reduced oxidative stress induced by H₂O₂. Furthermore, PEITC restored impaired insulin-stimulated glucose uptake, translocation of glucose transporter 4 and insulin signaling by H₂O₂. These results indicate that PEITC protected insulin-regulated glucose metabolism impaired by oxidative stress through the antioxidative activity in 3T3-L1 adipocytes.     

Differential expression and anti-oxidant function of glutathione peroxidase 3 in mouse uterus during decidualization

Glutathione peroxidase 3 (GPX3) is an important member of antioxidant enzymes for reducing reactive oxygen species and maintaining the oxygen balance. Gpx3 mRNA is strongly expressed in decidual cells from days 5 to 8 of pregnancy. After pregnant mice are treated with GPX inhibitor for 3 days, pregnancy rate is significantly reduced. Progesterone stimulates Gpx3 expression through PR/HIF1α in mouse endometrial stromal cells. In the decidua, the high level of GPX3 expression is closely associated with the reduction of hydrogen peroxide (H₂O₂). Based on our data, GPX3 may play a major role in reducing H₂O₂ during decidualization.     

Catalase is required for peroxisome maintenance during adipogenesis

Although obesity contributes to the onset and pathogenesis of metabolic diseases, it has been repeatedly demonstrated that being overweight or mildly obese carries a survival advantage compared with being thin or normal-weight. This relationship is called the obesity paradox. Hence, it is necessary to clarify the underlying mechanism of obesity onset for the prevention and treatment of these diseases. Catalase is distributed in peroxisomes under normal redox conditions and catalase activity is increased during the differentiation of 3T3-L1 preadipocytes to adipocytes. Although peroxisomes are responsible for lipid metabolism, the role of peroxisomal catalase in the process of lipid accumulation remains unclear. The present study aimed to investigate the relationships among catalase activity, peroxisome content, and lipid accumulation during the differentiation of 3T3-L1 preadipocytes to adipocytes. Increased catalase activity and lipid accumulation were observed during the differentiation of preadipocytes. Silencing of catalase by small interfering RNA or treatment with 3-amino-1,2,4-triazole (3-AT), a catalase inhibitor, resulted in reduced lipid accumulation. Inhibition of catalase activity in peroxisomes increases hydrogen peroxide (H₂O₂) levels, which results in a reduction of peroxisome content. Extracellular H₂O₂ had no influence on lipid accumulation during differentiation. The occurrence of autophagy was clearly enhanced in cells treated with 3-AT. Spautin-1, an inhibitor of autophagy flux, protected against a reduction in lipid accumulation by treatment with 3-AT. Our data provide evidence that catalase protects against the degradation of peroxisomes via the occurrence of autophagy triggered by the generation of H₂O₂ in peroxisomes. These results suggest that catalase in peroxisomes is crucial to adipogenesis.     

Low-Dose Bisphenol-A Impairs Adipogenesis and Generates Dysfunctional 3T3-L1 Adipocytes

Environmental endocrine disruptors (EDCs), including bisphenol-A (BPA), have been recently involved in obesity and diabetes by dysregulating adipose tissue function. Our aim was to examine whether prolonged exposure to low doses of BPA could affect adipogenesis and adipocyte metabolic functions. Therefore, 3T3-L1 pre-adipocytes were cultured for three weeks with BPA 1nM to mimic human environmental exposure. We evaluated BPA effect on cell proliferation, differentiation, gene expression and adipocyte metabolic function. BPA significantly increased pre-adipocyte proliferation (p<0.01). In 3T3-L1 adipocytes differentiated in the presence of BPA, the expression of Peroxisome proliferator-activated receptor gamma (PPARγ), Fatty Acid Binding Protein 4/Adipocyte Protein 2 (FABP4/AP2) and CCAAT/enhancer binding protein (C/EBPα) was increased by 3.5, 1.5 and 3 folds, respectively. Mature adipocytes also showed a significant increase in lipid accumulation (p<0.05) and alterations of insulin action, with significant reduction in insulin-stimulated glucose utilization (p<0.001). Moreover, in mature adipocytes, mRNA levels of Leptin, interleukin-6 (IL6) and interferon-γ (IFNγ) were significantly increased (p<0.05). In conclusion, BPA prolonged exposure at low doses, consistent with those found in the environment, may affect adipocyte differentiation program, enhancing pre-adipocyte proliferation and anticipating the expression of the master genes involved in lipid/glucose metabolism. The resulting adipocytes are hypertrophic, with impaired insulin signaling, reduced glucose utilization and increased pro-inflammatory cytokine expression. Thus, these data supported the hypothesis that BPA exposure, during critical stages of adipose tissue development, may cause adipocyte metabolic dysfunction and inflammation, thereby increasing the risk of developing obesity-related diseases.     

Nuclear Receptor Liver X Receptor Is O-GlcNAc-modified in Response to Glucose

Post-translational modification of nucleocytoplasmic proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) has for the last 25 years emerged as an essential glucose-sensing mechanism. The liver X receptors (LXRs) function as nutritional sensors for cholesterol-regulating lipid metabolism, glucose homeostasis, and inflammation. LXRs are shown to be post-translationally modified by phosphorylation, acetylation, and sumoylation, affecting their target gene specificity, stability, and transactivating and transrepressional activity, respectively. In the present study, we show for the first time that LXRα and LXRβ are targets for glucose-hexosamine-derived O-GlcNAc modification in human Huh7 cells. Furthermore, we observed increased hepatic LXRα O-GlcNAcylation in vivo in refed mice and in streptozotocin-induced refed diabetic mice. Importantly, induction of LXRα O-GlcNAcylation in both mouse models was concomitant with increased expression of the lipogenic gene SREBP-1c (sterol regulatory element-binding protein 1c). Furthermore, glucose increased LXR/retinoic acid receptor-dependent activation of luciferase reporter activity driven by the mouse SREBP-1c promoter via the hexosamine biosynthetic pathway in Huh7 cells. Altogether, our results suggest that O-GlcNAcylation of LXR is a novel mechanism by which LXR acts as a glucose sensor affecting LXR-dependent gene expression, substantiating the crucial role of LXR as a nutritional sensor in lipid and glucose metabolism.     

Sustained Submicromolar H2O2 Levels Induce Hepcidin via Signal Transducer and Activator of Transcription 3 (STAT3)

The peptide hormone hepcidin regulates mammalian iron homeostasis by blocking ferroportin-mediated iron export from macrophages and the duodenum. During inflammation, hepcidin is strongly induced by interleukin 6, eventually leading to the anemia of chronic disease. Here we show that hepatoma cells and primary hepatocytes strongly up-regulate hepcidin when exposed to low concentrations of H₂O₂ (0.3–6 μm), concentrations that are comparable with levels of H₂O₂ released by inflammatory cells. In contrast, bolus treatment of H₂O₂ has no effect at low concentrations and even suppresses hepcidin at concentrations of >50 μm. H₂O₂ treatment synergistically stimulates hepcidin promoter activity in combination with recombinant interleukin-6 or bone morphogenetic protein-6 and in a manner that requires a functional STAT3-responsive element. The H₂O₂-mediated hepcidin induction requires STAT3 phosphorylation and is effectively blocked by siRNA-mediated STAT3 silencing, overexpression of SOCS3 (suppressor of cytokine signaling 3), and antioxidants such as N-acetylcysteine. Glycoprotein 130 (gp130) is required for H₂O₂ responsiveness, and Janus kinase 1 (JAK1) is required for adequate basal signaling, whereas Janus kinase 2 (JAK2) is dispensable upstream of STAT3. Importantly, hepcidin levels are also increased by intracellular H₂O₂ released from the respiratory chain in the presence of rotenone or antimycin A. Our results suggest a novel mechanism of hepcidin regulation by nanomolar levels of sustained H₂O₂. Thus, similar to cytokines, H₂O₂ provides an important regulatory link between inflammation and iron metabolism.     

Hydrogen peroxide alters splicing of soluble guanylyl cyclase and selectively modulates expression of splicing regulators in human cancer cells

Background

Soluble guanylyl cyclase (sGC) plays a central role in nitric oxide (NO)-mediated signal transduction in the cardiovascular, nervous and gastrointestinal systems. Alternative RNA splicing has emerged as a potential mechanism to modulate sGC expression and activity. C-α1 sGC is an alternative splice form that is resistant to oxidation-induced protein degradation and demonstrates preferential subcellular distribution to the oxidized environment of endoplasmic reticulum (ER).

Methodology/Principal Findings

Here we report that splicing of C-α1 sGC can be modulated by H₂O₂ treatment in BE2 neuroblastoma and MDA-MD-468 adenocarcinoma human cells. In addition, we show that the H₂O₂ treatment of MDA-MD-468 cells selectively decreases protein levels of PTBP1 and hnRNP A2/B1 splice factors identified as potential α1 gene splicing regulators by in silico analysis. We further demonstrate that down-regulation of PTBP1 by H₂O₂ occurs at the protein level with variable regulation observed in different breast cancer cells.

Conclusions/Significance

Our data demonstrate that H₂O₂ regulates RNA splicing to induce expression of the oxidation-resistant C-α1 sGC subunit. We also report that H₂O₂ treatment selectively alters the expression of key splicing regulators. This process might play an important role in regulation of cellular adaptation to conditions of oxidative stress.     

The Arabidopsis Prohibitin Gene PHB3 Functions in Nitric Oxide–Mediated Responses and in Hydrogen Peroxide–Induced Nitric Oxide Accumulation

To discover genes involved in nitric oxide (NO) metabolism, a genetic screen was employed to identify mutants defective in NO accumulation after treatment with the physiological inducer hydrogen peroxide. In wild-type Arabidopsis thaliana plants, NO levels increase eightfold in roots after H₂O₂ treatment for 30 min. A mutant defective in H₂O₂-induced NO accumulation was identified, and the corresponding mutation was mapped to the prohibitin gene PHB3, converting the highly conserved Gly-37 to an Asp in the protein''s SPFH domain. This point mutant and a T-DNA insertion mutant were examined for other NO-related phenotypes. Both mutants were defective in abscisic acid–induced NO accumulation and stomatal closure and in auxin-induced lateral root formation. Both mutants were less sensitive to salt stress, showing no increase in NO accumulation and less inhibition of primary root growth in response to NaCl treatment. In addition, light-induced NO accumulation was dramatically reduced in cotyledons. We found no evidence for impaired H₂O₂ metabolism or signaling in the mutants as H₂O₂ levels and H₂O₂-induced gene expression were unaffected by the mutations. These findings identify a component of the NO homeostasis system in plants and expand the function of prohibitin genes to include regulation of NO accumulation and NO-mediated responses.     

Regulatory functions of ROS dynamics via glutathione metabolism and glutathione peroxidase activity in developing rice zygote

Reactive oxygen species (ROS) play essential roles in plant development and environmental stress responses. In this study, ROS dynamics, the glutathione redox status, the expression and subcellular localization of glutathione peroxidases (GPXs), and the effects of inhibitors of ROS-mediated metabolism were investigated along with fertilization and early zygotic embryogenesis in rice (Oryza sativa). Zygotes and early embryos exhibited developmental arrest upon inhibition of ROS production. Egg cells accumulated high ROS levels, and, after fertilization, intracellular ROS levels progressively declined in zygotes in which de novo expression of GPX1 and 3 was observed through upregulation of the genes. In addition to inhibition of GPX activity, depletion of glutathione impeded early embryonic development and led to failure of the zygote to appropriately decrease H₂O₂ levels. Moreover, through monitoring of the glutathione redox status, the developing zygotes exhibited a progressive glutathione oxidation, which became extremely delayed under inhibited GPX activity. Our results provide insights into the importance of ROS dynamics, GPX antioxidant activity, and glutathione redox metabolism during zygotic/embryonic development.     

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.     

Synthesis and kinetic evaluation of a trifunctional enzyme mimic with a dimanganese active centre

Glutathione peroxidase (GPX), superoxide dismutase (SOD) and catalase (CAT) play crucial roles in the metabolism and homeostasis of reactive oxygen species (ROS) in living organisms. From examination of the steady state and pre-steady state kinetic behavior of natural GPX it was found that, in contrast to accepted theories, the affinity of the enzyme for H₂O₂ rather than reduced glutathione (GSH) most significantly affects its kinetic behavior. Consequently, an enzyme mimic was produced with a similar affinity for the substrate H₂O₂. A salicylaldehyde Schiff base containing a dimanganese centre was selected as a precursor, because it has high H₂O₂-binding affinity for such a relatively small molecule and similar catalytic activity to that of SOD and CAT. Selenium was also incorporated into the catalytic center to provide activity similar to that of GPX, and thus trifunctional enzymatic activity. The K_mH2O2 of the mimic (7.32 × 10^-2 mM) was found quite close to that of natural enzyme (1.0 × 10^-2 mM), indicating that the affinity of the mimic to H₂O₂ was successfully increased to approach natural GPX. The steady state kinetic performance of the enzyme mimic showed that the ratio between k_cat/K_mGSH and k_cat/ K_mH2O2 was quite similar to that of native GPX, indicating that the Mn(III)₂(L-Se-SO₃Na) had the same selectivity for both substrates GSH and H₂O₂ as native GPX, which put it among the best existing GPX mimics. Moreover, the new mimic was confirmed to strongly inhibit lipid peroxidation and mitochondrial swelling, probably due to the synergism between the three antioxidant enzymatic activities.     

Decreased Cystathionine-γ-lyase (CSE) Activity in Livers of Type 1 Diabetic Rats and Peripheral Blood Mononuclear Cells (PBMC) of Type 1 Diabetic Patients

The liver plays a major role in the formation of H₂S, a novel signaling molecule. Diabetes is associated with lower blood levels of H₂S. This study investigated the activities of cystathionine-γ-lyase (CSE, the enzyme that catalyzes H₂S formation) in livers of type 1 diabetic (T1D) animals and in peripheral blood mononuclear cells (PBMC) isolated from T1D patients. T1D is associated with both hyperketonemia (acetoacetate and β-hydroxybutyrate) and hyperglycemia. This study also examined the role of hyperglycemia and hyperketonemia per se in decreased CSE activity using U937 monocytes and PBMC isolated from healthy subjects. Livers from streptozotocin-treated T1D rats demonstrated a significantly higher reactive oxygen species production, lower CSE protein expression and activity, and lower H₂S formation compared with those of controls. Studies with T1D patients showed a decrease in CSE protein expression and activity in PBMC compared with those of age-matched normal subjects. Cell culture studies demonstrated that high glucose (25 mm) and/or acetoacetate (4 mm) increased reactive oxygen species, decreased CSE mRNA expression, protein expression, and enzymatic activity, and reduced H₂S levels; however, β-hydroxybutyrate treatment had no effect. A similar effect, which was also observed in PBMC treated with high glucose alone or along with acetoacetate, was prevented by vitamin D supplementation. Studies with CSE siRNA provide evidence for a relationship between impaired CSE expression and reduced H₂S levels. This study demonstrates for the first time that both hyperglycemia and hyperketonemia mediate a reduction in CSE expression and activity, which can contribute to the impaired H₂S signaling associated with diabetes.     

Hydrogen Sulfide Promotes Adipogenesis in 3T3L1 Cells

The effect of hydrogen sulfide (H₂S) on differentiation of 3T3L1-derived adipocytes was examined. Endogenous H₂S was increased after 3T3L1 differentiation. The expression of the H₂S-synthesising enzymes, cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST), was increased in a time-dependent manner during 3T3L1 differentiation. Expression of genes associated with adipogenesis related genes including fatty acid binding protein 4 (FABP4/aP2), a key regulator of this process, was increased by GYY4137 (a slow-releasing H₂S donor compound) and sodium hydrosulfide (NaHS, a classical H₂S donor) but not by ZYJ1122 or time-expired NaHS. Furthermore expression of these genes were reduced by aminooxyacetic acid (AOAA, CBS inhibitor), DL-propargylglycine (PAG, CSE inhibitor) as well as by CSE small interference RNA (siCSE) and siCBS. The size and number of lipid droplets in mature adipocytes was significantly increased by both GYY4137 and NaHS, which also impaired the ability of CL316,243 (β3-agonist) to promote lipolysis in these cells. In contrast, AOAA and PAG had the opposite effect. Taken together, we show that the H₂S-synthesising enzymes CBS, CSE and 3-MST are endogenously expressed during adipogenesis and that both endogenous and exogenous H₂S modulate adipogenesis and adipocyte maturation.     

Regulation of ascorbate peroxidase activity in dark-grown radish cotyledons by a catalase inhibitor, 3-Amino-1,2,4-Triazole

The present study was performed to see the physiological role of cytosolic ascorbate peroxidase (APX) and its relationship to other enzymes involved in the H₂O₂ scavenging metabolism, and also to elucidate the regulation of APX expression in dark-grown radish (Raphanus sativus L. cv Taiwang) cotyledons. To do so, 3-amino-l,2,4-triazole (aminotriazole), a known specific inhibitor of catalase, was used to simulate a catalase-deficient phenomenon in cotyledons. Aminotriazole, in very low concetration (10^-4 M), inhibited remarkably the development of catalase activity in cotyledons during dark germination. This inhibition of catalase by aminotriazole, however, did not result in any significant changes in the growth response and the H₂O₂ level of developing cotyledons. In addition, the development of guaiacol peroxidase (GPX) activity was also not significantly affected. Unlike GPX, cytosolic APX activity was induced rapidly and reached a 1.7-fold increase in aminotriazole treated cotyledons at day 7 after germination. However,in vitro incubation of cytosolic APX preparation from cotyledons with aminotriazole did not result in any significant change in activity. One cytosolic APX isozyme (APXa) band involved in this APX activation was predominantly intensified in a native polyacrylamide gel by activity staining assay. This means that this APXa isozyme seems to play a key role in the expression of cytosolic APX activity. On the other hand, 2-day-old control seedlings treated with exogenous 1 mM H₂O₂ for 1 h showed a significant increase of cytosolic APX acitivity even in the absence of aminotriazole. Also, 2 μM cycloheximide treatment substantially inhibited the increase of APX activity due to aminotriazole. Based on these results, we suggest that a radish cytosolic APX could probably be substituted for catalase in H₂O₂ removal and that the expression of APX seems to be regulated by a change of endogenous H₂O₂ level which couples to APX protein synthesis in a translation stage in cotyledons.     

Molecular identification for epigallocatechin-3-gallate-mediated antioxidant intervention on the H2O2-induced oxidative stress in H9c2 rat cardiomyoblasts

Background

Epigallocatechin-3-gallate (EGCG) has been documented for its beneficial effects protecting oxidative stress to cardiac cells. Previously, we have shown the EGCG-mediated cardiac protection by attenuating reactive oxygen species and cytosolic Ca²⁺ in cardiac cells during oxidative stress and myocardial ischemia. Here, we aimed to seek a deeper elucidation of the molecular anti-oxidative capabilities of EGCG in an H₂O₂-induced oxidative stress model of myocardial ischemia injury using H9c2 rat cardiomyoblasts.

Results

Proteomics analysis was used to determine the differential expression of proteins in H9c2 cells cultured in the conditions of control, 400 μM H₂O₂ exposure for 30 min with and/or without 10 to 20 μM EGCG pre-treatment. In this model, eight proteins associated with energy metabolism, mitochondrial electron transfer, redox regulation, signal transduction, and RNA binding were identified to take part in EGCG-ameliorating H₂O₂-induced injury in H9c2 cells. H₂O₂ exposure increased oxidative stress evidenced by increases in reactive oxygen species and cytosolic Ca²⁺ overload, increases in glycolytic protein, α-enolase, decreases in antioxidant protein, peroxiredoxin-4, as well as decreases in mitochondrial proteins, including aldehyde dehydrogenase-2, ornithine aminotransferase, and succinate dehydrogenase ubiquinone flavoprotein subunit. All of these effects were reversed by EGCG pre-treatment. In addition, EGCG attenuated the H₂O₂-induced increases of Type II inositol 3, 4-bisphosphate 4-phosphatase and relieved its subsequent inhibition of the downstream signalling for Akt and glycogen synthase kinase-3β (GSK-3β)/cyclin D1 in H9c2 cells. Pre-treatment with EGCG or GSK-3β inhibitor (SB 216763) significantly improved the H₂O₂-induced suppression on cell viability, phosphorylation of pAkt (S473) and pGSK-3β (S9), and level of cyclin D1 in cells.

Conclusions

Collectively, these findings suggest that EGCG blunts the H₂O₂-induced oxidative effect on the Akt activity through the modulation of PIP3 synthesis leading to the subsequent inactivation of GSK-3β mediated cardiac cell injury.