Tetsuo Adachi

Supplementation of hydrogen-rich water improves lipid and glucose metabolism in patients with type 2 diabetes or impaired glucose tolerance.

Oxidative stress is recognized widely as being associated with various disorders including diabetes, hypertension, and atherosclerosis. It is well established that hydrogen has a reducing action. We therefore investigated the effects of hydrogen-rich water intake on lipid and glucose metabolism in patients with either type 2 diabetes mellitus (T2DM) or impaired glucose tolerance (IGT). We performed a randomized, double-blind, placebo-controlled, crossover study in 30 patients with T2DM controlled by diet and exercise therapy and 6 patients with IGT. The patients consumed either 900 mL/d of hydrogen-rich pure water or 900 mL of placebo pure water for 8 weeks, with a 12-week washout period. Several biomarkers of oxidative stress, insulin resistance, and glucose metabolism, assessed by an oral glucose tolerance test, were evaluated at baseline and at 8 weeks. Intake of hydrogen-rich water was associated with significant decreases in the levels of modified low-density lipoprotein (LDL) cholesterol (ie, modifications that increase the net negative charge of LDL), small dense LDL, and urinary 8-isoprostanes by 15.5% (P < .01), 5.7% (P < .05), and 6.6% (P < .05), respectively. Hydrogen-rich water intake was also associated with a trend of decreased serum concentrations of oxidized LDL and free fatty acids, and increased plasma levels of adiponectin and extracellular-superoxide dismutase. In 4 of 6 patients with IGT, intake of hydrogen-rich water normalized the oral glucose tolerance test. In conclusion, these results suggest that supplementation with hydrogen-rich water may have a beneficial role in prevention of T2DM and insulin resistance.

Ethanol Extract of Brazilian Red Propolis Induces Apoptosis in Human Breast Cancer MCF-7 Cells through Endoplasmic Reticulum Stress

Propolis, a natural product collected from plants by honey bees, is commonly used in folk medicines. Endoplasmic reticulum (ER) stress is known to induce apoptosis through the induction of CCAAT/enhancer-binding protein homologous protein (CHOP). Here, we investigated whether ethanol extracts of propolis and caffeic acid phenethyl ester (CAPE) induce apoptosis, mitochondrial dysfunction, and ER stress in human breast cancer MCF-7 cells and human fibroblasts. Among several ethanol extracts of propolis and CAPE, Brazilian red propolis (BRP) significantly reduced MCF-7 cell viability through the induction of mitochondrial dysfunction, caspase-3 activity, and DNA fragmentation but did not affect those of fibroblasts. Moreover, treatment with BRP significantly induced CHOP expression in MCF-7 cells compared to fibroblasts. Further, pretreatment with a chemical chaperone, 4-phenylbutyric acid, suppressed BRP-triggered MCF-7 cell death. Overall, we revealed that an ethanol extract of BRP induces MCF-7 cell apoptosis through, at least in part, ER stress-related signaling.

Cobalt chloride decreases EC-SOD expression through intracellular ROS generation and p38-MAPK pathways in COS7 cells.

It is known that cells suffer a chronic hypoxic condition during the development of proximal tubulointerstitial disease. However, it is accepted that extracellular-superoxide dismutase (EC-SOD) protects the cells from oxidative stress. The purpose of this study was to elucidate the regulation of EC-SOD expression in cells under hypoxia. The results show that the expressions of EC-SOD mRNA and protein in cobalt chloride (CoCl2)-treated COS7 cells decreased in a dose- and time-dependent manner, whereas the expressions of other SOD isoforms (Cu/Zn-SOD and Mn-SOD) were not changed. The down-regulation of EC-SOD mRNA was suppressed by pre-treatment with the antioxidant trolox and the p38 mitogen-activated protein kinase (p38-MAPK) inhibitor SB203580. It is concluded that the expression of EC-SOD is decreased through ROS and p38-MAPK signalling cascades and that the down-regulation of EC-SOD leads to a decrease in the resistance to oxidative stress of COS7 cells under hypoxia induced by CoCl2.

Endoplasmic reticulum stress inducers provide protection against 6-hydroxydopamine-induced cytotoxicity

6-Hydroxydopamine (6-OHDA) is a neurotoxin used to establish experimental models of Parkinsons disease. Exposure to 6-OHDA results in cell death associated with oxidative stress. Pretreatments with sublethal oxidative stress and some pharmacological drugs have been shown to exert preconditioning effects on cytotoxicity caused by 6-OHDA. In this study, we investigated whether endoplasmic reticulum (ER) stress exerts preconditioning effects on 6-OHDA-induced cytotoxicity in human neuroblastoma SH-SY5Y cells. Pretreatment with ER stress inducers, thapsigargin (Tg) and tunicamycin (Tm), promoted GRP78 mRNA induction and ATF4 translation, which are ER stress markers, under our experimental conditions and protected against the cytotoxicity. The protective effect of Tg was more potent than that of Tm. We also found that Tg induced the expression of the antioxidant gene heme oxygenase-1 (HO-1) in a dose-dependent manner, whereas Tm had a weak effect on HO-1 induction. Flow cytometric analysis revealed that reactive oxygen species generated by 6-OHDA were more effectively suppressed in cells pretreated with Tg than with Tm. Therefore, it is likely that Tg enhances antioxidative defenses in SH-SY5Y cells compared with Tm. Because actinomycin D inhibited HO-1 induction by Tg, the induction of HO-1 may be regulated at the transcriptional level. Moreover, the specific eIF2α phosphatase inhibitor salubrinal augmented Tg-induced HO-1 expression. Therefore, the downstream signaling pathway of eIF2α might be involved in Tg-induced HO-1 expression. On the other hand, the reporter assay revealed that Tg stimulated the antioxidant response element (ARE) that is located in regulatory regions of antioxidant genes such as HO-1. Taken together, our data suggest that preconditioning effects induced by Tg mediate an adaptive response to 6-OHDA-induced cytotoxicity via phosphorylation of eIF2α and activation of the ARE.

Contribution of p38 MAPK, NF-κB and glucocorticoid signaling pathways to ER stress-induced increase in retinal endothelial permeability.

Diabetic retinopathy (DR) is characterized by the development of intraretinal microvascular abnormalities. Endoplasmic reticulum (ER) stress is known to play a pathogenic role in vascular impairment in DR. The present study demonstrated that the treatment of human retinal endothelial cells with ER stress inducers such as thapsigargin (Tg) and tunicamycin (Tm) significantly increased the permeability of exogenously added FITC-dextran, accompanied by a decrease of transendothelial electrical resistance (TEER). The expression of claudin-5 among tight junction proteins was significantly decreased by the treatment with Tg or Tm. A p38 MAPK inhibitor, SB203580, and an NF-κB inhibitor, dexamethasone, significantly suppressed the Tg-induced down-regulation of claudin-5, decrease of TEER and leakage of added FITC-dextran. The translocation of NF-κB p65 subunit to the nucleus was also inhibited by the addition of SB203580 or dexamethasone. The effects of dexamethasone are thought to be due to the transrepression of the above signaling and direct regulation of claudin-5 gene.

Extracellular-superoxide dismutase expression during monocytic differentiation of U937 cells

Leukemic cell lines, such as U937, THP‐1, and HL60 cells, can differentiate into macrophages following exposure to various agents including 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) in vitro. It is well known that TPA enhances reactive oxygen species (ROS) generation through the activation of NADPH oxidase (NOX), and ROS act as mediators in TPA signaling. Extracellular‐superoxide dismutase (EC‐SOD) is a major anti‐oxidative enzyme that protects the cells from damaging effects of superoxide. Recently, the reduction of Cu/Zn‐SOD and the induction of Mn‐SOD by TPA in leukemic cells have been reported; however, the regulation of EC‐SOD by TPA remains poorly understood. Here, we explored the regulation of EC‐SOD during the monocytic differentiation of U937 cells by TPA. We observed the reduction of EC‐SOD and Cu/Zn‐SOD, whereas the induction of Mn‐SOD during the differentiation of U937 cells. The reduction of EC‐SOD and Cu/Zn‐SOD was attenuated by pretreatments with GF109203X (an inhibitor of protein kinase C, PKC), diphenyleneiodonium (an inhibitor of NOX), and U0126 (an inhibitor of mitogen‐activated protein kinase kinase, MEK/extracellular‐signal regulated kinase, ERK). Interestingly, pretreatment with BAY11‐7082 (an inhibitor of nuclear factor‐κB, NF‐κB) suppressed the reduction of Cu/Zn‐SOD, but not of EC‐SOD. Furthermore, we also determined the involvement of newly synthesized protein and the instability of mRNA in the reduction of EC‐SOD. Overall, our results suggest that the expression of EC‐SOD is decreased by TPA through intracellular signaling consisting of PKC, NOX‐derived ROS and MEK/ERK, but not of NF‐κB signaling. J. Cell. Biochem. 112: 244–255, 2011.

Expression of extracellular superoxide dismutase during adipose differentiation in 3T3-L1 cells

Abstract Obesity is known to be the primary causal component in metabolic syndrome. Adipocytes in obese patients exhibit increased oxidative stress via the activation of reactive oxygen species (ROS)-producing systems and inactivation of antioxidant enzymes. Extracellular superoxide dismutase (EC-SOD) is an anti-inflammatory enzyme that protects cells from the damaging effects of ROS. An earlier report showed that plasma EC-SOD levels in type 2 diabetic patients were significantly and inversely related to body mass index and homeostasis model assessment-insulin resistance index. Moreover, the administration of pioglitazone, an antidiabetic agent, significantly increased the plasma level of EC-SOD. In this report, the expression of EC-SOD was compared to other adipocytokines in mice 3T3-L1 pre-adipocytes. EC-SOD expression levels were increased after the induction of differentiation and then declined, which was similar to adiponectin and transcription factors such as peroxisome proliferator-activated receptor-γ (PPAR-γ) and CCAAT/enhancer-binding protein-α (C/EBP-α). On the other hand, the expression levels of pro-inflammatory adipocytokines, such as tumor necrosis factor-α (TNF-α) and monocyte chemo-attractant protein-1 (MCP-1), increased markedly in the development stage of cells. It was observed that the expression of EC-SOD in differentiated 3T3-L1 cells co-cultured with LPS-stimulated J774 macrophages was up-regulated, while the addition of TNF-α down-regulated EC-SOD and adiponectin expression in adipocytes. It is known that infiltrated and activated macrophages produce extracellular ROS at high levels in adipose tissue. It is possible that the expression of EC-SOD in adipocytes was stimulated to protect them from oxidative stress in the co-culture system.

Olmesartan improves endothelial function in hypertensive patients: link with extracellular superoxide dismutase

Endothelial dysfunction in essential hypertension is an independent predictor for future cardiovascular events. Although inhibition of the renin-angiotensin system (RAS) reportedly improves endothelial function through its effects on oxidative stress and inflammation, questions remain regarding the factors that are pivotal for improvement of endothelial function by RAS inhibition. We therefore performed a prospective, randomized crossover trial in which an angiotensin II type 1 receptor antagonist, olmesartan and calcium channel blocker, amlodipine, were compared in 31 essential hypertensive patients. Results showed that, although both treatments achieved comparable lowering of blood pressure (BP), olmesartan, but not amlodipine, significantly improved endothelial function as evaluated by flow-mediated vasodilation (FMD) in the brachial artery. Although no significant changes in diabetic and lipid parameters were observed with either drug, olmesartan slightly decreased estimated glomerular filtration rate, which, surprisingly, translated into decreased microalbuminuria. In a similar vein, olmesartan reduced serum C-reactive protein and increased urine antioxidant levels compared with baseline, and reduced urine 8-epi-prostaglandin F2α levels compared with both baseline and amlodipine. Finally, although overall changes in plasma extracellular superoxide dismutase (EC-SOD) levels were not modulated by either treatment, for olmesartan there was a positive correlation between changes in FMD and those in EC-SOD levels. In conclusion, olmesartan improved endothelial function in hypertensive patients independent of its BP-lowering effect, which was due, at least in part, to its antioxidative property. Therefore, olmesartan might provide a greater long-term benefit for hypertensive patients with impaired endothelial function than amlodipine.

The effect of hypoxia mimetic cobalt chloride on the expression of EC-SOD in 3T3-L1 adipocytes

Abstract It is well known that adipose tissue is not only a passive reservoir for energy storage but also produces and secretes a variety of bioactive molecules called adipocytokines, including adiponectin and tumor necrosis factor-α (TNF-α). Recently, it has been reported that adipose tissue can suffer a chronic hypoxic condition during hypertrophy of adipocytes, and this condition leads to the dysregulation of adipocytokines. Further, hypoxic adipocytes are in an increased oxidative stress. Extracellular-superoxide dismutase (EC-SOD) is an anti-inflammatory enzyme that protects cells from reactive oxygen species (ROS) by scavenging superoxide anion. Previous reports showed that plasma EC-SOD levels in type 2 diabetes patients were significantly and inversely related to the body mass index, homeostasis model assessment-insulin resistance index; however, the mechanisms of EC-SOD and adiponectin reductions during hypoxia remain poorly understood. Here, we demonstrate that cobalt chloride (CoCl2), a hypoxia mimetic, decreases EC-SOD and adiponectin in 3T3-L1 adipocytes by intracellular ROS-independent, but TNF-α and c-jun N-terminal kinase (JNK)-dependent mechanisms. From these results, it is possible that TNF-α is a key regulator of the reduction of EC-SOD and adiponectin in CoCl2-treated 3T3-L1 adipocytes, and we speculated that the reduction of EC-SOD and adiponectin would lead to and/or promote metabolic disorders.

Epigenetic regulation of extracellular-superoxide dismutase in human monocytes.

Extracellular-superoxide dismutase (EC-SOD) is a major SOD isozyme mainly present in the vascular wall and plays an important role in normal redox homeostasis. We previously showed the significant reduction or induction of EC-SOD during human monocytic U937 or THP-1 cell differentiation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), respectively; however, its cell-specific expression and regulation have not been fully elucidated. It has been reported that epigenetic factors, such as DNA methylation and histone modification, are involved in several kinds of gene regulation. In this study, we investigated the involvement of epigenetic factors in EC-SOD expression and determined high levels of DNA methylation within promoter and coding regions of EC-SOD in THP-1 cells compared to those in U937 cells. Moreover, treatment with a DNA methyltransferase inhibitor, 5-azacytidine, significantly induced the expression of EC-SOD in THP-1 cells, indicating the importance of DNA methylation in the suppression of EC-SOD expression; however, the DNA methylation status did not change during THP-1 cell differentiation induced by TPA. On the other hand, we detected histone H3 and H4 acetylation during differentiation. Further, pretreatment with histone acetyltransferase inhibitors, CPTH2 or garcinol, significantly suppressed the TPA-inducible EC-SOD expression. We also determined the epigenetic suppression of EC-SOD in peripheral blood mononuclear cells. Treatment with granulocyte macrophage colony-stimulating factor (GM-CSF)/granulocyte-CSF induced that expression. Overall, these findings provide novel evidence that cell-specific and TPA-inducible EC-SOD expression are regulated by DNA methylation and histone H3 and H4 acetylation in human monocytic cells.