Nanako Itoh

Application of water-soluble radical initiator, 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride, to a study of oxidative stress.

It is essential to generate free radicals at a controled and constant rate for specific duration and at specific site to study the dynamics of oxidation and also antioxidation. Both hydrophilic and lipophilic azo compounds have been used for such purpose. In the present work, the action of 2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH) was examined and compared with those of 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) and 2,2′-azobis[2-methyl-N-(2-hydroxyethyl)-propionamide] (AMHP). The rate constant of free radical formation (ekd) for AIPH was 2.6 × 10-6/s at 37°C in PBS (pH 7.4) solution, indicating that AIPH gives 3.8 times more free radicals than AAPH under the same conditions. It was found that the dynamics of oxidation and antioxidation induced by AIPH can be studied satisfactorily in the oxidation in micelles, LDL and erythrocyte suspensions, plasma, and cultured cells. The extent of cell death induced by AIPH and AAPH was directly proportional to the total free radicals formed. Interestingly, it was found that rats would not drink water containing AAPH, but they drank water containing AIPH. The levels of 8-iso-prostaglandin F2α (8-isoPs), 7-hydroxycholesterol (FCOH), lysophosphatidylcholine in the plasma of rats given water containing 50 mM AIPH for 1 month increased compared with those of control rats which drank water without AIPH. It may be concluded that AIPH is useful for kinetic and mechanistic studies on oxidative stress to membranes, lipoproteins, cells, and even animal models.

Wheat Alkylresorcinols Suppress High-Fat, High-Sucrose Diet-Induced Obesity and Glucose Intolerance by Increasing Insulin Sensitivity and Cholesterol Excretion in Male Mice

BACKGROUND Epidemiologic studies have shown that the consumption of whole grains can reduce the risk of type 2 diabetes mellitus, cardiovascular disease, and all-cause mortality. However, the underlying mechanisms remain a matter of debate. OBJECTIVE We aimed to determine the effects of wheat bran-derived alkylresorcinols on diet-induced metabolic disorders in mice. METHODS We fed C57BL/6J mice a normal refined diet or a high-fat, high-sucrose diet [29.1% fat, 20.7% protein, 34.0% carbohydrates containing 20.0% sucrose (w/w)] alone (FS) or containing 0.4% (wt:wt) alkylresorcinols (FS-AR) for 10 wk. RESULTS The alkylresorcinols suppressed FS-induced increases in body weight by 31.0% as well as FS-induced hepatic triglyceride accumulation (means ± SEMs: 29.6 ± 3.18 and 19.8 ± 2.42 mg/g tissue in the FS and FS-AR groups, respectively), without affecting energy intake. We measured circadian changes in blood metabolic hormones and found that FS-induced hyperinsulinemia (5.1 and 2.1 μg/L at night in the FS and FS-AR groups, respectively) and hyperleptinemia (21.6 and 10.8 μg/L at night in the FS and FS-AR groups, respectively) were suppressed by alkylresorcinols. Glucose and insulin tolerance tests showed that alkylresorcinols significantly reduced fasting blood glucose concentrations (190 ± 3.62 and 160 ± 8.98 mg/dL in the FS and FS-AR groups, respectively) and suppressed glucose intolerance as well as insulin resistance induced by the FS diet. Furthermore, alkylresorcinols significantly increased insulin-stimulated hepatic serine/threonine protein kinase B phosphorylation compared to the FS diet (+81.3% and +57.4% for Ser473 and Thr308, respectively). On the other hand, pyruvate and starch tolerance tests suggested that alkylresorcinols did not affect gluconeogenesis and carbohydrate digestion, respectively. Alkylresorcinols significantly increased fecal cholesterol excretion by 39.6% and reduced blood cholesterol concentrations by 30.4%, while upregulating the expression of hepatic cholesterol synthetic genes such as sterol regulatory element binding protein 2 (Srebf2) and 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (Hmgcs1). CONCLUSIONS These findings suggest that wheat alkylresorcinols increase glucose tolerance and insulin sensitivity by suppressing hepatic lipid accumulation and intestinal cholesterol absorption, which subsequently suppresses diet-induced obesity in mice.

Distribution of tocopherols and tocotrienols to rat ocular tissues after topical ophthalmic administration

With increasing evidence suggesting the involvement of oxidative stress in various disorders and diseases, the role of antioxidants in vivo has received much attention. Chemically, tocopherols and tocotrienols are closely related; however, it has been observed that they have widely varying degrees of biological effectiveness. The present study has been carried out in an attempt to deepen our understanding of whether there is a significant difference in distribution between tocopherol and tocotrienol homologs to rat eye tissues. Rats were administered 5 μl of pure tocopherol or tocotrienol to each eye once a day for 4 d. Various tissues of the eyes were separated and analyzed for tocopherol and tocotrienol concentrations. The concentration of α-tocotrienol increased markedly in every tissue to which it was administered; however, no significant increase was observed in the case of α-tocopherol. The intraocular penetration of γ-tocopherol and γ-tocotrienol did not differ significantly. Additionally, a significant increase in total vitamin E concentration was observed in ocular tissues, including crystalline lens, neural retina, and eye cup, with topical administration using a relatively small amount (5 μL) of vitamin E, whereas no significant increase was observed when the same amount of vitamin E was administered orally. Topical administration of tocotrienols is thus an effective way to increase ocular tissue vitamin E concentration.

Continuous Exposure to a Novel Stressor Based on Water Aversion Induces Abnormal Circadian Locomotor Rhythms and Sleep-Wake Cycles in Mice

Psychological stressors prominently affect diurnal rhythms, including locomotor activity, sleep, blood pressure, and body temperature, in humans. Here, we found that a novel continuous stress imposed by the perpetual avoidance of water on a wheel (PAWW) affected several physiological diurnal rhythms in mice. One week of PAWW stress decayed robust circadian locomotor rhythmicity, while locomotor activity was evident even during the light period when the mice are normally asleep. Daytime activity was significantly upregulated, whereas nighttime activity was downregulated, resulting in a low amplitude of activity. Total daily activity gradually decreased with increasing exposure to PAWW stress. The mice could be exposed to PAWW stress for over 3 weeks without adaptation. Furthermore, continuous PAWW stress enhanced food intake, but decreased body weight and plasma leptin levels, indicating that sleep loss and PAWW stress altered the energy balance in these mice. The diurnal rhythm of corticosterone levels was not severely affected. The body temperature rhythm was diurnal in the stressed mice, but significantly dysregulated during the dark period. Plasma catecholamines were elevated in the stressed mice. Continuous PAWW stress reduced the duration of daytime sleep, especially during the first half of the light period, and increased nighttime sleepiness. Continuous PAWW stress also simultaneously obscured sleep/wake and locomotor activity rhythms compared with control mice. These sleep architecture phenotypes under stress are similar to those of patients with insomnia. The stressed mice could be entrained to the light/dark cycle, and when they were transferred to constant darkness, they exhibited a free-running circadian rhythm with a timing of activity onset predicted by the phase of their entrained rhythms. Circadian gene expression in the liver and muscle was unaltered, indicating that the peripheral clocks in these tissues remained intact.

Disrupted daily light-dark cycle induces the expression of hepatic gluconeogenic regulatory genes and hyperglycemia with glucose intolerance in mice.

We elucidated associations between metabolic disorders and the environmental light-dark (LD) cycle that entrains the circadian clock located in the suprachiasmatic nucleus of mammals. Mice were fed with a high-fat/high-sucrose diet for eight weeks under a normal 12h light-12h dark cycle (LD 12:12) or an ultradian 3h light-3h dark cycle (LD 3:3) that might perturb the central clock. The circadian behavioral rhythms were gradually disturbed under LD 3:3. Hyperglycemia with glucose intolerance and increases in diabetic markers, glycated albumin and hemoglobin A1c, were significantly induced without affecting body weight gain and food consumption in LD 3:3. Expression levels of hepatic gluconeogenic regulatory genes such as Pck1, G6pc, Hnf4a, and Foxo1/3/4 genes were increased under LD 3:3. Hypercholesterolemia with hepatic cholesterol accumulation was also induced in LD 3:3. Ultradian LD 3:3 cycles did not affect the adipose inflammation that is considered a major player in obesity-associated metabolic disorders. Our findings provide a link between metabolic disorders and environmental photoperiodic cycles in genetically normal animals.

The role of α-tocopherol in motor hypofunction with aging in α-tocopherol transfer protein knockout mice as assessed by oxidative stress biomarkers.

It has been hypothesized that oxidative stress plays a key role in aging. In order to elucidate the role of the antioxidant network - including alpha-tocopherol (alphaT) and alphaT transfer protein - in aging in vivo, alpha-tocopherol transfer protein knockout (alphaTTP(-/-)) mice were fed a vitamin-E-depleted diet, and wild-type (WT) mice were fed a diet containing 0.002 wt.% alphaT from the age of 3 months to 1 1/2 years. The lipid oxidation markers total hydroxyoctadecadienoic acid (tHODE) and 8-iso-prostaglandin F(2)alpha, and antioxidant levels in the blood, liver and brain were measured at 3, 6, 12 and 18 months. tHODE levels in the plasma of alphaTTP(-/-) mice were elevated at 6 months compared to 3 months, and were significantly higher those in WT mice, although they decreased thereafter. On the other hand, tHODE levels in the liver and brain were constantly higher in alphaTTP(-/-) mice than in WT mice. Motor activities decreased with aging in both mouse types; however, those in the alphaTTP(-/-) mice were lower than those in the WT mice. It is intriguing to note that motor activities were significantly correlated with the stereoisomer ratio (Z,E/E,E) of HODE, which is a measure of antioxidant capacity in vivo, in the plasma, in the liver and even in the brain, but not with other factors such as antioxidant levels. In summary, using the biomarker tHODE and its stereoisomer ratio, we demonstrated that alphaT depletion was associated with a decrease in motor function, and that this may be primarily attributable to a decrease in the total antioxidant capacity in vivo.

Low-Carbohydrate, High-Protein Diet Affects Rhythmic Expression of Gluconeogenic Regulatory and Circadian Clock Genes in Mouse Peripheral Tissues

Recent studies have demonstrated that metabolic changes in mammals induce feedback regulation of the circadian clock. The present study evaluates the effects of a low-carbohydrate high-protein diet (HPD) on circadian behavior and peripheral circadian clocks in mice. Circadian rhythms of locomotor activity and core body temperature remained normal in mice fed with the HPD diet (HPD mice), suggesting that it did not affect the central clock in the hypothalamus. Two weeks of HPD feeding induced mild hypoglycemia without affecting body weight, although these mice consumed more calories than mice fed with a normal diet (ND mice). Plasma insulin levels were increased during the inactive phase in HPD mice, but increased twice, beginning and end of the active phase, in ND mice. Expression levels of the key gluconeogenic regulatory genes PEPCK and G6Pase were significantly induced in the liver and kidneys of HPD mice. The HPD appeared to induce peroxisome proliferator-activated receptor α (PPARα) activation, since mRNA expression levels of PPARα and its typical target genes, such as PDK4 and Cyp4A10, were significantly increased in the liver and kidneys. Circadian mRNA expression of clock genes, such as BMAL1, Cry1, NPAS2, and Rev-erbα, but not Per2, was significantly phase-advanced, and mean expression levels of BMAL1 and Cry1 mRNAs were significantly elevated, in the liver and kidneys of HPD mice. These findings suggest that a HPD not only affects glucose homeostasis, but that it also advances the molecular circadian clock in peripheral tissues. (Author correspondence: k-ooishi@aist.go.jp)

Dietary heat-killed Lactobacillus brevis SBC8803 promotes voluntary wheel-running and affects sleep rhythms in mice

AIMS We previously reported that heat-killed Lactobacillus brevis SBC8803 enhances appetite via changes in autonomic neurotransmission. Here we assessed whether a diet supplemented with heat-killed SBC8803 affects circadian locomotor rhythmicity and sleep architecture. MAIN METHODS AND KEY FINDINGS Daily total activity gradually increased in mice over 4 weeks and supplementation with heat-killed SBC8803 significantly intensified the increase, which reached saturation at 25 days. Electroencephalography revealed that SBC8803 supplementation significantly reduced the total amount of time spent in non-rapid eye movement (NREM) sleep and increased the amount of time spent being awake during the latter half of the nighttime, but tended to increase the total amount of time spent in NREM sleep during the daytime. Dietary supplementation with SBC8803 can extend the duration of activity during the nighttime and of sleep during the daytime. Daily voluntary wheel-running and sleep rhythmicity become intensified when heat-killed SBC8803 is added to the diet. SIGNIFICANCE Dietary heat-killed SBC8803 can modulate circadian locomotion and sleep rhythms, which might benefit individuals with circadian rhythms that have been disrupted by stress or ageing.

Effects of intraduodenal injection of Lactobacillus brevis SBC8803 on autonomic neurotransmission and appetite in rodents

Lactobacilli provide several health benefits to mammals, including humans. We previously observed that in rats, intraduodenal injection of Lactobacillus johnsonii La1 elevated efferent gastric vagal nerve activity (efferent-GVNA), while Lactobacillus paracasei ST11 suppressed efferent-GVNA, and thereby increased or decreased food intake. To determine the function of Lactobacillus brevis (SBC8803), its effect on food intake was examined by providing food containing heat-killed SBC8803 to mice. We observed that administration of SBC8803 elevated food intake. Because the afferent intestinal vagal nerve (IVN) is hypothesized to be involved in efferent-GVNA changes, we examined the effect of intraduodenal administration of heat-killed SBC8803 on efferent-GVNA and afferent-IVN activity (IVNA) in rats. In this study, we found that intraduodenal administration of heat-killed SBC8803 increased both efferent-GVNA and afferent-IVNA in rats. Moreover, IV administration of the serotonin 3 receptor antagonist granisetron eliminated the effects of SBC8803 on efferent-GVNA and afferent-IVNA. These findings suggest that heat-killed SBC8803 enhances appetite by elevating digestion and absorption abilities via changes in autonomic neurotransmission that might be mediated by the serotonin 3 receptor.

Disruption of behavioral circadian rhythms induced by psychophysiological stress affects plasma free amino acid profiles without affecting peripheral clock gene expression in mice.

Disordered circadian rhythms are associated with various psychiatric conditions and metabolic diseases. We recently established a mouse model of a psychophysiological stress-induced chronic sleep disorder (CSD) characterized by reduced amplitude of circadian wheel-running activity and sleep-wake cycles, sleep fragmentation and hyperphagia. Here, we evaluate day-night fluctuations in plasma concentrations of free amino acids (FAA), appetite hormones and prolactin as well as the hepatic expression of circadian clock-related genes in mice with CSD (CSD mice). Nocturnal increases in wheel-running activity and circadian rhythms of plasma prolactin concentrations were significantly disrupted in CSD mice. Hyperphagia with a decreased leptin/ghrelin ratio was found in CSD mice. Day-night fluctuations in plasma FAA contents were severely disrupted without affecting total FAA levels in CSD mice. Nocturnal increases in branched-chain amino acids such as Ile, Leu, and Val were further augmented in CSD mice, while daytime increases in Gly, Ala, Ser, Thr, Lys, Arg, His, Tyr, Met, Cys, Glu, and Asn were significantly attenuated. Importantly, the circadian expression of hepatic clock genes was completely unaffected in CSD mice. These findings suggest that circadian clock gene expression does not always reflect disordered behavior and sleep rhythms and that plasma FFA profiles could serve as a potential biomarker of circadian rhythm disorders.