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.

Loss of Mitochondrial DNA by Gemcitabine Triggers Mitophagy and Cell Death.

Gemcitabine (2,2-difluorodeoxycytidine nucleic acid), an anticancer drug exhibiting a potent ability to kill cancer cells, is a frontline chemotherapy drug. Although some chemotherapeutic medicines are known to induce nuclear DNA damage, no investigation into mitochondrial DNA (mtDNA) damage currently exists. When we treated insulinoma pancreatic ß-cells (line INS-1) with high mitochondrial activity with gemcitabine for 24 h, the mtDNA contents were decreased. Gemcitabine induced a decrease in the number of mitochondria and the average potential of mitochondrial membrane in the cell but increased the superoxide anion radical levels. We observed that treatment with gemcitabine to induce cell death accompanied by autophagy-related protein markers, Atg5 and Atg7; these were significantly prevented by the autophagy inhibitors. The localization of Atg5 co-occurred with the location of mitochondria with membranes having high potential and mitophagy in cells treated with gemcitabine. The occurrence of mitophagy was inhibited by the inhibitors of the phosphatidylinositol 3-kinase/Akt pathway. Our results led us to the conclusion that gemcitabine induced cell death through mitophagy with the loss of mtDNA. These findings may provide a rationale for the combination of mtDNA damage with mitophagy in future clinical applications for cancer cells.

[Linezolid-induced Apoptosis through Mitochondrial Damage and Role of Superoxide Dismutase-1 in Human Monocytic Cell Line U937].

Cytopenia is a major adverse event associated with linezolid therapy. The objective of this study was to examine whether the cytotoxicity of linezolid to eukaryotic cells was associated with mitochondrial dysfunction and apoptosis-like cell death in human leukemic monocyte lymphoma cell line U937. Apoptosis-like cell death was clearly observed when cells were incubated with linezolid, depending on the duration and linezolid concentration. Mitochondrial membrane potential of cells treated with linezolid collapsed in a short period of time, but the number of mitochondria did not decrease. Cytotoxicity of linezolid was relieved by the knockdown of superoxide dismutase-1 in U937 cells. On the other hand, no autophagy was observed in cells treated with linezolid. These results suggest that mitochondrial damages would be linked to the induction of apoptosis in U937 cells treated with linezolid and that its mechanism does not involve autophagy.

Increase of Anti-oxidative Capacity during Differentiation of 3T3-L1 Preadipocytes into Adipocytes.

Cells have developed ingenious defense mechanisms in response to oxidative stress. Here, we evaluated changes in anti-oxidative capacity during differentiation of 3T3-L1 preadipocytes into adipocytes. When 3T3-L1 preadipocytes were treated with H2O2 (0.10-2.0 mM) for 21 h, cell viability decreased in response to H2O2 concentration, with an LD50 of approximately 0.35 mM H2O2. In the cells undergoing differentiation at 2 and 6 d, LD50 increased to 1.0 and >2.0 mM H2O2, respectively. These results indicate that resistance to oxidative stress dramatically increased with progression of differentiation of preadipocytes into adipocytes. Catalase activity and GSH content increased in the differentiated cells at 6 d, whereas superoxide dismutase and glutathione peroxidase activities were slightly lower in adipocytes than in preadipocytes. Moreover, knockdown of catalase or depletion of intracellular GSH enhanced the sensitivity to H2O2. When GSH was added to the cells depleted of intracellular GSH, the antioxidant capacity recovered. Autophagy was increased in differentiated adipocytes but was not affected by H2O2 treatment. Therefore, these results suggest that the increase in intracellular catalase activity and GSH content played a role in the increased anti-oxidative capacity of differentiated 3T3-L1 adipocytes.

A randomized, placebo-controlled, double-blind clinical trial of rikkunshito for patients with non-erosive reflux disease refractory to proton-pump inhibitor: the G-PRIDE study.

BACKGROUND: The aim of this study was to investigate the efficacy of rikkunshito (RKT), a traditional Japanese medicine, combined with proton pump inhibitor (PPI) in patients with PPI-refractory non-erosive reflux disease (NERD). METHODS: Patients with PPI-refractory NERD (n = 242) were randomly assigned to the RKT group [rabeprazole (10 mg/day) + RKT (7.5 g/t.i.d.) for 8 weeks] or the placebo group (rabeprazole + placebo). After the 4- and 8-week treatments, we assessed symptoms and quality of life (QOL) using the Frequency Scale for the Symptoms of Gastroesophageal Reflux Disease (FSSG), Gastrointestinal Symptom Rating Scale (GSRS), and Short-Form Health Survey-8 (SF-8). RESULTS: There were no significant differences in FSSG and GSRS score improvement between these groups after the 4- and 8-week treatments. The mental component summary (MCS) scores of the SF-8 improved more in the RKT group (from 45.8 ± 8.1 to 48.5 ± 7.4) than in the placebo group (from 47.7 ± 7.1 to 48.4 ± 7.5) after the 4-week treatment (P < 0.05). The 8-week treatment with RKT was more effective for improvement of the degree of MCS score in patients with a low body mass index (<22) (P < 0.05) and significantly improved the acid-related dysmotility symptoms of FSSG in female and elderly patients (≥ 65 years). CONCLUSION: There were no significant differences in improvement of GERD symptoms in patients with PPI-refractory NERD between these groups. However, RKT may be useful for improving mental QOL in non-obese patients and acid-related dyspeptic symptoms, especially in women and the elderly.

Impact of vancomycin or linezolid therapy on development of renal dysfunction and thrombocytopenia in Japanese patients.

BACKGROUND: Vancomycin and linezolid therapies are associated with renal dysfunction and thrombocytopenia, respectively. METHODS: We retrospectively investigated Japanese patients with renal dysfunction or thrombocytopenia possibly associated with vancomycin and linezolid therapies, including 235 patients treated with parenteral vancomycin and 178 treated with parenteral linezolid. RESULTS: Renal dysfunction occurred more frequently in patients receiving vancomycin (24%) than in those receiving linezolid (13%; p = 0.032), whereas thrombocytopenia occurred more frequently in linezolid-treated patients (41%) than in vancomycin-treated patients (17%; p < 0.001). Controlling trough vancomycin concentrations (<20 µg/ml) protects against renal dysfunction, but thrombocytopenia may occur after >7.5 days of linezolid treatment. CONCLUSION: Controlling trough vancomycin concentrations to <20 µg/ml protects Japanese patients against renal dysfunction. Linezolid is an appropriate initial therapy for severe infections in patients with acute renal dysfunction, but monitoring of platelet counts is essential after initiation of therapy.

Involvement of phosphatidylinositol 3-kinase/Akt pathway in gemcitabine-induced apoptosis-like cell death in insulinoma cell line INS-1.

This study demonstrated gemcitabine-induced cytotoxicity in the insulinoma cell line INS-1. Gemcitabine inhibited INS-1 cell proliferation and maintained consistent cell number for 24 h, and then caused apoptosis within 48 h of incubation. Since gemcitabine activates the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway, which is involved in the resistance of pancreatic exocrine cancer to gemcitabine, we investigated the participation of this pathway in gemcitabine-induced cytotoxicity in INS-1 cells. LY294002 and wortmannin, two PI3-K inhibitors, significantly prevented gemcitabine-induced cytotoxicity in INS-1 cells, indicating that the PI3-K/Akt pathway is involved in gemcitabine-induced cytotoxicity. Gemcitabine-induced Akt phosphorylation in INS-1 cells was prevented by LY294002. Although gemcitabine induced cell cycle arrest at the G1 and early S phases, LY294002 did not inhibit the cell cycle. These data suggest that PI3-K activation does not influence gemcitabine-induced cell cycle arrest. In gemcitabine-treated cells, nuclear fragmentation and DNA ladder formation were observed. These findings suggest that gemcitabine induced apoptotic cell death in INS-1 cells through the activation of the PI3-K/Akt pathway.

Hydrogen peroxide induces cell cycle arrest in cardiomyoblast H9c2 cells, which is related to hypertrophy.

Cell cycle arrest is associated with differentiation, senescence and apoptosis. We investigated alterations in the cell cycle during the development of hypertrophy induced by hydrogen peroxide (H(2)O(2)) in the H9c2 clonal myoblastic cell line. H(2)O(2) induced hypertrophy in H9c2 cells that was indicated by an increase in atrial natriuretic peptide (ANP) gene expression, a marker of cardiomyocyte hypertrophy, and a larger cell size. On induction of hypertrophy by H(2)O(2) in H9c2 cells, cell proliferation was arrested, indicated by the number of cells remaining constant during a 72-h incubation period. The cell cycle was arrested at the G1 and G2/M phases with an increase in p21 expression, a negative cell cycle regulator. Cell cycle arrest and increase in p21 expression were significantly inhibited by 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM), an intracellular calcium chelator. Although ANP gene expression was induced significantly, H(2)O(2) failed to induce hypertrophy in the presence of BAPTA-AM, and the cell cycle progressed. We concluded that H(2)O(2) induced cell cycle arrest in H9c2 cells, which was related to cellular hypertrophy.

Gastric volvulus as a complication in the recipients after adult living donor liver transplantation.

BACKGROUND: We report 4 adult cases of mesenteroaxial gastric volvulus after living donor liver transplantation (LDLT). RESULTS: All 4 recipients were female with a median age of 31 years (range, 21-69). All had undergone right lobe LDLT. Gastric volvulus developed on postoperative days (POD) 4-30, and all were successfully treated with an endoscopic correction procedure. Two of 4 needed a repeated correction procedure and 1 needed a surgical revision for the recurrent volvulus. CONCLUSION: Although this type of the complication is unusual, earlier post-transplant endoscopic intervention is useful to reverse the pyloroantral obstruction. These cases let us recognize that gastric volvulus is one of the complications after right lobe LDLT.

Cardiomyocyte H9c2 Cells Exhibit Differential Sensitivity to Intracellular Reactive Oxygen Species Generation with Regard to Their Hypertrophic vs Death Responses to Exogenously Added Hydrogen Peroxide.

Many researchers have hypothesized that differences in reactive oxygen species levels can trigger the cellular decision between hypertrophy and cell death in cardiomyocytes. In the present study, we examined the relationship between reactive oxygen species levels and hypertrophy or cell death in H9c2 cardiomyocytes after the addition of hydrogen peroxide. Following addition of hydrogen peroxide, we observed a slight increase in fluorescence intensity of 2',7'-dichlorofluorescein, a probe of intracellular reactive oxygen species, and cell hypertrophy in H9c2 cells (normal cells). In contrast, a dramatic increase in fluorescence intensity was followed by cell death in glutathione-depleted H9c2 cells. In the presence of the antioxidant Trolox or the iron chelator deferoxamine, both normal and glutathione-depleted cells developed hypertrophy without a concomitant increase in levels of reactive oxygen species. An inhibitor of p53, pifithrin-alpha, prevented cell death after the addition of hydrogen peroxide; instead a substantial increase in levels of reactive oxygen species and hypertrophy were observed. These results suggest that H9c2 cells exhibit differential sensitivity to intracellular reactive oxygen species generation with regard to their hypertrophic versus death responses to exogenously added hydrogen peroxide.

Necrotic pathway in human osteosarcoma Saos-2 cell death induced by chloroacetaldehyde.

Chloroacetaldehyde, a metabolite of the anticancer drug ifosfamide, may be responsible for serious adverse effects like encephalopathy in ifosfamide chemotherapy. In this study, we demonstrate that chloroacetaldehyde, but not ifosfamide, induces cell death in human osteosarcoma Saos-2 cells and we investigated the mechanism by which this occurs. Chloroacetaldehyde above 30 micromol/l induced significant cell death in a time-dependent manner. Thiol compounds such as N-acetyl cysteine, glutathione and dithiothreitol protected the cells against chloroacetaldehyde-induced cell death, although other nonthiol compounds and the antioxidative enzymes superoxide dismutase and catalase did not, suggesting that reactive oxygen species might not mediate cell death. In cells exposed to chloroacetaldehyde, levels of both total thiols and glutathione were significantly reduced. Chloroacetaldehyde also collapsed the mitochondrial membrane potential of these cells, induced the release of cytochrome c from mitochondria to the cytosol and significantly reduced cellular ATP levels during the course of death. The mitochondrial potential collapse was also prevented by thiol compounds. Flow cytometric analyses by means of annexin-V and propidium iodide double staining and immunofluorescence staining of active caspase-3 revealed that cells subjected to a lethal dose of chloroacetaldehyde displayed features characteristic of necrosis and that caspase-3 was not activated in response to chloroacetaldehyde. Taken together, these findings suggest that Saos-2 cells exposed to chloroacetaldehyde die by necrosis resulting from a decrease in intracellular thiols, disruption of the mitochondrial membrane potential and the depletion of cellular ATP.

Ascorbate-mediated iron release from ferritin in the presence of alloxan.

Release of iron from ferritin requires reduction of ferric to ferrous iron. The iron can participate in the diabetogenic action of alloxan. We investigated the ability of ascorbate to catalyze the release of iron from ferritin in the presence of alloxan. Incubation of ferritin with ascorbate alone elicited iron release (33 nmol/10 min) and the generation of ascorbate free radical, suggesting a direct role for ascorbate in iron reduction. Iron release by ascorbate significantly increased in the presence of alloxan, but alloxan alone was unable to release measurable amounts of iron from ferritin. Superoxide dismutase significantly inhibited ascorbate-mediated iron release in the presence of alloxan, whereas catalase did not. The amount of alloxan radical (A.(-)) generated in reaction systems containing both ascorbate and alloxan decreased significantly upon addition of ferritin, suggesting that A.(-) is directly involved in iron reduction. Although release of iron from ferritin and generation of A.(-) were also observed in reactions containing GSH and alloxan, the amount of iron released in these reactions was not totally dependent on the amount of A.(-) present, suggesting that other reductants in addition to A.(-) (such as dialuric acid) may be involved in iron release mediated by GSH and alloxan. These results suggest that A.(-) is the main reductant involved in ascorbate-mediated iron release from ferritin in the presence of alloxan and that both dialuric acid and A.(-) contribute to GSH/alloxan-mediated iron release.

Requirement of intracellular free thiols for hydrogen peroxide-induced hypertrophy in cardiomyocytes.

Reactive oxygen species (ROS) are by-products of aerobic metabolism and are implicated in the pathogenesis of several diseases. H(2)O(2) produces oxidative stress and acts as a second messenger in several cell types. We tested whether the effect of H(2)O(2) on cellular events could be altered by changes in the intracellular redox status in a cardiomyocyte cell line. Using flow cytometric measurements, we found that adding H(2)O(2) induced hypertrophy in control cells in a time-dependent manner. Pre-incubation of the cells with buthionine sulfoximine (BSO), an inhibitor of de novo GSH synthesis, induced increase in the number of cells of small sizes by the addition of H(2)O(2) as compared to non-BSO pre-incubated control cells, and exacerbated the decrease in viability. Total thiol and GSH levels in H9c2 cells pre-incubated with BSO were about 75 and 30% of control, respectively, and GSH levels fell to below the limitation of detection after the addition of H(2)O(2), although total thiol levels were not markedly decreased. In the cells pre-incubated with BSO, hypertrophy was not observed by the addition of H(2)O(2) at any level of concentration. N-acetyl-L-cysteine and cysteine not only prevented increase in the number of cells of small sizes caused by H(2)O(2) but also induced hypertrophy in cells pre-incubated with BSO. These results suggest that the intracellular free thiol levels determine whether cell death or hypertrophy occurs in cardiomyocytes in the presence of H(2)O(2). On the other hand, the hypertrophied cells did not become larger by adding H(2)O(2), but had high levels of cellular GSH, suggesting the possibility that the hypertrophied cells have tolerance to oxidative stress.

[Anesthetic management of a patient with Evans syndrome--benefit of perioperative hemodilutional autologous blood transfusion].

The combination of idiopathic thrombocytopenic purpura and autoimmune hemolytic anemia is termed Evans syndrome. We experienced the anesthetic management of a patient with this syndrome undergoing laparoscopic splenectomy. After induction of general anesthesia using thiamylal, fentanyl and vecuronium, hemodilutional autologous transfusion was employed to minimize the immune hemolytic process against the transfused blood throughout the surgical procedure. The anesthetic course was uneventful, and neither massive hemorrhage nor any complications related to bleeding occurred. We therefore recommend the use of hemodilutional autologous transfusion for the anteshetic management of patients with Evans syndrome.

[Alloxan radical-induced generation of reactive oxygen species in the reaction system of alloxan with ascorbate].

The diabetogenic action of alloxan is thought to be initiated by generation of reactive oxygen species (ROS). Ascorbate can be an antioxidant in a predominantly aqueous environment, such as plasma and extracellular fluids. We have investigated the generation of ROS in the interaction of alloxan with ascorbate. Rapid oxygen consumption was observed in the reactin system of alloxan with ascorbate. The oxygen consumption was suppressed by superoxide dismutase and catalase, suggesting that superoxide and hydrogen peroxide could be generated in the reaction system. In addition, the generation of alloxan radical, an electron reductance of alloxan, and ascorbate free radical (AFR), an electron oxidant of ascorbate, was observed using electron spin resonance (ESR). Under anaerobic conditions, the ESR signal intensity of alloxan radical was significantly increased in comparison with that under aerobic conditions, whereas the intensity of AFR was significantly decreased. These results suggest that alloxan radical and AFR were generated in the reaction system of alloxan with ascorbate, and that the alloxan radical but not AFR reacted with molecular oxygen, resulting in the generation of ROS.