Kazutoshi Murotomi

Antioxidative and Antidiabetic Effects of Natural Polyphenols and Isoflavones

Many polyphenols that contain more than two phenolic hydroxyl groups are natural antioxidants and can provide health benefits to humans. These polyphenols include, for example, oleuropein, hydroxytyrosol, catechin, chlorogenic acids, hesperidin, nobiletin, and isoflavones. These have been studied widely because of their strong radical-scavenging and antioxidative effects. These effects may contribute to the prevention of diseases, such as diabetes. Insulin secretion, insulin resistance, and homeostasis are important factors in the onset of diabetes, a disease that is associated with dysfunction of pancreatic β-cells. Oxidative stress is thought to contribute to this dysfunction and the effects of antioxidants on the pathogenesis of diabetes have, therefore, been investigated. Here, we summarize the antioxidative effects of polyphenols from the perspective of their radical-scavenging activities as well as their effects on signal transduction pathways. We also describe the preventative effects of polyphenols on diabetes by referring to recent studies including those reported by us. Appropriate analytical approaches for evaluating antioxidants in studies on the prevention of diabetes are comprehensively reviewed.

Chemistry of Lipid Peroxidation Products and Their Use as Biomarkers in Early Detection of Diseases

We developed a novel method to measure hydroxyoctadecadienoic acid (HODE) levels in biological fluids and tissue samples. This method can be used to measure the oxidation products of linoleic acid. Reduction and saponification enabled us to measure hydroperoxides and hydroxides of both free and esterified forms of linoleic acid as total HODE, which includes the enzymatic and non-enzymatic products 9- and 13-(Z, E)-HODEs; the non-enzymatic free radical-mediated products 9- and 13-(E, E)-HODEs; and the specific non-enzymatic singlet oxygen-mediated products 10- and 12-(Z, E)-HODEs. We have recently reported HODE levels in plasma and erythrocytes from healthy volunteers and patients with several diseases and determined that its levels are much higher in patients with lifestyle-related diseases than in healthy volunteers. Furthermore, 10- and 12-(Z, E)-HODE plasma levels can serve as promising biomarkers for the early detection of diabetes. Thus, HODE is a useful biomarker for the assessment of oxidative status, and its efficiency as a biomarker can be improved by using it in combination with other typical biomarkers. This review article focuses on lipid peroxidation biomarkers, including HODE, and discusses their potential in practical and clinical applications in disease prediction.

Oleuropein-Rich Diet Attenuates Hyperglycemia and Impaired Glucose Tolerance in Type 2 Diabetes Model Mouse

Oleuropein, a phenolic compound found in abundance in olive leaves, has beneficial effects on various diseases. However, it is unknown whether an oleuropein-rich diet is efficacious against type 2 diabetic phenotypes. In this study, we investigated the effects of the oleuropein-containing supplement OPIACE, whose oleuropein content exceeds 35% (w/w), on the diabetic phenotypes in type 2 diabetes model Tsumura Suzuki Obese Diabetes (TSOD) mouse. TSOD mice were fed OPIACE at 4 weeks of age, i.e., before the TSOD mice exhibited diabetic phenotypes. We revealed that OPIACE attenuated hyperglycemia and impaired glucose tolerance in TSOD mice over the long-term (from 10 to 24 weeks of age) but had no effect on obesity. Furthermore, we demonstrated that OPIACE mildly reduced oxidative stress in TSOD mice by 26.2% and attenuated anxiety-like behavioral abnormality in aged TSOD mice. The results suggest that oleuropein suppresses the progression of type 2 diabetes and diabetes-related behavioral abnormality over the long-term.

Radical-scavenging Activity and Antioxidative Effects of Olive Leaf Components Oleuropein and Hydroxytyrosol in Comparison with Homovanillic Alcohol

Olive leaf has great potential as a natural antioxidant, and one of its major phenolic components is oleuropein. In this study, the antioxidant activity of oleuropein against oxygen-centered radicals was measured by examining its sparing effects on the peroxyl radical-induced decay of fluorescein and pyrogallol red, in comparison with related compounds. The antioxidant capacity of oleuropein against lipid peroxidation was also assessed through its effect on the free radical-induced oxidation of methyl linoleate in a micelle system. On a molar basis, oleuropein and hydroxytyrosol inhibited the decay of fluorescein for longer than both homovanillic alcohol and the vitamin-E mimic 2-carboxy-2,5,7,8-tetramethyl-6-chromanol (Trolox), but did not suppress pyrogallol red decay in a concentration-dependent manner. Measurement of the fluorescein decay period revealed that the stoichiometric number of oleuropein and hydroxytyrosol against peroxyl radicals was twice that of Trolox, which is substantially higher than expectations based on chemical structure. Oleuropein and hydroxytyrosol were also more effective than Trolox and homovanillic alcohol at suppressing the oxidation of methyl linoleate in the micelle system. Thus, both oleuropein and hydroxytyrosol exhibit high antioxidative activity against lipid peroxidation induced by oxygen-centered radicals, but the high reactivity of phenolic/catecholic radicals makes their mechanism of action complex.

Switching from singlet-oxygen-mediated oxidation to free-radical-mediated oxidation in the pathogenesis of type 2 diabetes in model mouse.

Abstract Oxidative stress plays a key role in the development of type 2 diabetes. However, it is still unknown what kind of oxidative stress underlies the development of type 2 diabetes. We investigated hydroxyoctadecadienoic acid (HODE) isomers, which have been proposed as a biomarker for evaluating oxidative stress in vivo, during the development of diabetes in Tsumura Suzuki Obese Diabetes (TSOD) mouse, a type 2 diabetes model. It was revealed that glucose tolerance and insulin resistance index HOMA-IR in TSOD mice at 5 weeks of age were approximately normal, namely, the mice were in the prediabetic state, but these levels were significantly exacerbated from 8 weeks of age compared with those in Tsumura Suzuki Non Obesity (TSNO) mice (control). Concomitantly, the plasma levels of free-radical-mediated oxidation products, 9- and 13-(E,E)-HODE and 7β-hydroxycholesterol, in TSOD mice were significantly higher than those in TSNO mice at 8, and 8 and 11 weeks of age, respectively. Interestingly, the plasma levels of 10- and 12-(Z,E)-HODE, which are produced specifically by singlet-oxygen-mediated oxidation, in TSOD mice were higher than those in TSNO mice only at 5 weeks of age, and not at 8, 11, and 13 weeks of age. We demonstrated that singlet-oxygen-mediated oxidation occurred in TSOD mice before development of the diabetic phenotypes, including impaired glucose tolerance and insulin resistance. These results suggest that excessive singlet-oxygen-mediated oxidation plays an important role in the pathogenesis of type 2 diabetes.

Highly sensitive luciferase reporter assay using a potent destabilization sequence of calpain 3

Reporter assays that use luciferases are widely employed for monitoring cellular events associated with gene expression in vitro and in vivo. To improve the response of the luciferase reporter to acute changes of gene expression, a destabilization sequence is frequently used to reduce the stability of luciferase protein in the cells, which results in an increase of sensitivity of the luciferase reporter assay. In this study, we identified a potent destabilization sequence (referred to as the C9 fragment) consisting of 42 amino acid residues from human calpain 3 (CAPN3). Whereas the half-life of Emerald Luc (ELuc) from the Brazilian click beetle Pyrearinus termitilluminans was reduced by fusing PEST (t1/2=9.8 to 2.8h), the half-life of C9-fused ELuc was significantly shorter (t1/2=1.0h) than that of PEST-fused ELuc when measurements were conducted at 37°C. In addition, firefly luciferase (luc2) was also markedly destabilized by the C9 fragment compared with the humanized PEST sequence. These results indicate that the C9 fragment from CAPN3 is a much more potent destabilization sequence than the PEST sequence. Furthermore, real-time bioluminescence recording of the activation kinetics of nuclear factor-κB after transient treatment with tumor necrosis factor α revealed that the response of C9-fused ELuc is significantly greater than that of PEST-fused ELuc, demonstrating that the use of the C9 fragment realizes a luciferase reporter assay that has faster response speed compared with that provided by the PEST sequence.

Involvement of splenic iron accumulation in the development of nonalcoholic steatohepatitis in Tsumura Suzuki Obese Diabetes mice.

Nonalcoholic steatohepatitis (NASH) is a common hepatic manifestation of metabolic syndrome and can lead to hepatic cirrhosis and cancer. It is considered that NASH is caused by multiple parallel events, including abnormal lipid metabolism, gut-derived-endotoxin-induced inflammation, and adipocytokines derived from adipose tissue, suggesting that other tissues are involved in NASH development. Previous studies demonstrated that spleen enlargement is observed during the course of NASH pathogenesis. However, the involvement of splenic status in the progression of NASH remains unclear. In this study, we examined hepatic and splenic histopathological findings in the early stage of NASH using the Tsumura Suzuki Obese Diabetes (TSOD) mouse model established for assessing NASH. We found that 12-week-old TSOD mice clearly exhibited the histopathological features of NASH in the early stage. At this age, the spleen of TSOD mice showed markedly higher iron level than that of control Tsumura Suzuki Non Obesity (TSNO) mice. The level of accumulated iron was significantly decreased by feeding a diet with glucosyl hesperidin, a bioactive flavonoid, accompanied with alleviation of hepatic lesions. Furthermore, we found that splenic iron level was positively correlated with the severity of NASH manifestations, suggesting that abnormalities in the spleen are involved in the development of NASH.

Type 2 diabetes model TSOD mouse is exposed to oxidative stress at young age

Tsumura Suzuki Obese Diabetes (TSOD) mouse, a model of obese type 2 diabetes, older than around 11 weeks of age develops diabetic phenotypes. Previous studies have indicated that the development of diabetes is partly due to three loci associated with body weight and glucose homeostasis. However, little is known about the initial events triggering the development of the diabetic phenotypes in TSOD mouse. Here, we investigated the alteration of diabetes-related parameters, including the levels of blood glucose and inflammatory cytokines, and the oxidative stress status, in young TSOD mice. TSOD mice at 5 weeks of age showed increases in body weight and plasma total cholesterol level, but not hyperglycemia or impaired glucose tolerance, compared with age-matched control Tsumura Suzuki Non-Obese (TSNO) mice. Plasma tumor necrosis factor (TNF)-α and interleukin (IL)-6 were not detected in TSOD mice at 5 weeks of age. However, plasma total hydroxyoctadecadienoic acid (tHODE), a biomarker of oxidative stress, was increased in TSOD mice relative to TSNO mice at same age. The results demonstrated that young TSOD mice are exposed to oxidative stress before developing the diabetic phenotypes, and suggested that oxidative stress is an initial event triggering the development of diabetes in TSOD mice.

Antioxidant properties of 5-hydroxy-4-phenyl-butenolide via activation of Nrf2/ARE signaling pathway

5-Hydroxy-4-phenyl-butenolide (5H4PB) is a bioactive compound with antifungal and anti-obesity properties. Although it has recently been shown that 5H4PB activates peroxisome proliferator-activated receptor-gamma (PPARγ), the effect of 5H4PB on intracellular signaling pathways has not been clarified. In this study, we found that 5H4PB activated the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway, which plays an important role in cellular defense against oxidative stress, and the subsequent upregulation of ARE-dependent cytoprotective genes, including the heme oxygenase-1, catalase, and superoxide dismutase genes, without exhibiting cytotoxicity. In addition, 5H4PB significantly attenuated intracellular ROS generation, glutathione oxidation, and DNA damage induced by hydrogen peroxide (H2O2) exposure in mouse fibroblast cells. Furthermore, we demonstrated that pretreatment with 5H4PB confers a significant cytoprotective effect against H2O2-induced cell death in mouse cultured fibroblasts and primary hepatocytes. Thus, our study demonstrated that 5H4PB enhanced cellular resistance to oxidative damage via activation of the Nrf2/ARE signaling pathway.

Comparative analysis of the intestinal flora in type 2 diabetes and nondiabetic mice

A relationship between type 2 diabetes mellitus (T2DM) and intestinal flora has been suggested since development of analysis technology for intestinal flora. An animal model of T2DM is important for investigation of T2DM. Although there are some animal models of T2DM, a comparison of the intestinal flora of healthy animals with that of T2DM animals has not yet been reported. The intestinal flora of Tsumura Suzuki Obese Diabetes (TSOD) mice was compared with that of Tsumura, Suzuki, Non Obesity (TSNO) mice in the present study. The TSOD mice showed typical type 2 diabetes symptoms, which were high-fat diet-independent. The TSOD and the TSNO mouse models were derived from the same strain, ddY. In this study, we compared the intestinal flora of TSOD mice with that if TSNO mice at 5 and 12 weeks of age. We determined that that the number of operational taxonomic units (OTUs) was significantly higher in the cecum of TSOD mice than in that of TSNO mice. The intestinal flora of the cecum and that of the feces were similar between the TSNO and the TSOD strains. The dominant bacteria in the cecum and feces were of the phyla Firmicutes and Bacteroidetes. However, the content of some bacterial species varied between the two strains. The percentage of Lactobacillus spp. within the general intestinal flora was higher in TSOD mice than in TSNO mice. In contrast, the percentages of order Bacteroidales and family Lachnospiraceae were higher in TSNO mice than in TSOD mice. Some species were observed only in TSOD mice, such as genera Turicibacter and SMB53 (family Clostridiaceae), the percentage of which were 3.8% and 2.0%, respectively. Although further analysis of the metabolism of the individual bacteria in the intestinal flora is essential, genera Turicibacter and SMB53 may be important for the abnormal metabolism of type 2 diabetes.