Endogenous Androgens Diminish Food Intake and Activation of Orexin A Neurons in Response to Reduced Glucose Availability in Male Rats.

Sex steroids modify feeding behavior and body weight regulation, and androgen reportedly augments food intake and body weight gain. To elucidate the role of endogenous androgens in the feeding regulation induced by reduced glucose availability, we examined the effect of gonadectomy (orchiectomy) on food intake and orexin A neuron's activity in the lateral hypothalamic/perifornical area (LH/PFA) in response to reduced glucose availability (glucoprivation) induced by 2-deoxy-d-glucose (2DG) administration in male rats. Rats (7W) were bilaterally orchiectomized (ORX group) or sham operated (Sham group). Seventeen days after the surgery, food intake response to 2DG (400 mg/kg, i.v.) was measured for 4 h after the infusion. The same experiment was performed for the immunohistochemical examination of c-Fos-expressing orexin A neurons in the LH/PFA and c-Fos expression in the arcuate nucleus (Arc). Food intake induced by glucoprivation was greater in the ORX group than the Sham group, and the glucoprivation-induced food intake was inversely correlated with plasma testosterone concentration. Glucoprivation stimulated c-Fos expression of the orexin A neurons at the LH/PFA and c-Fos expression in the dorsomedial Arc. The number and percentage of c-Fos-expressing orexin A neurons in the LH/PFA and c-Fos expression in the dorsomedial Arc were significantly higher in the ORX group than the Sham group. This indicates that endogenous androgen, possibly testosterone, diminishes the food intake induced by reduced glucose availability, possibly via the attenuated activity of orexin A neuron in the LH/PFA and neurons in the dorsomedial Arc.

Fluoxetine Mimics the Anorectic Action of Estrogen and Its Regulation of Circadian Feeding in Ovariectomized Female Rats.

Our previous study demonstrated that chronic estrogen replacement in ovariectomized rats reduces food intake and augments c-Fos expression in the suprachiasmatic nucleus (SCN), specifically during the light phase. Here, we hypothesized that serotonergic neurons in the central nervous system (CNS), which have anorectic action and play a role in regulating circadian rhythm, mediate the light phase-specific anorectic action of estrogen, and that selective serotonin reuptake inhibitors (SSRIs) mimic the hypophagic action of estrogen. Female Wistar rats were ovariectomized and treated with estradiol (E2) or cholesterol by subcutaneously implanting a silicon capsule containing E2 or cholesterol. Then, half of the cholesterol-treated rats were injected with the SSRI fluoxetine (5 mg/kg) (FLX group), while the remaining rats in the cholesterol-treated group (CON group) and all those in the E2 group were injected with saline subcutaneously twice daily at the onsets of the light and dark phases. Both E2 and FLX reduced food intake during the light phase but not the dark phase, and reduced body weight gain. In addition, both E2 and FLX augmented the c-Fos expression in the SCN, specifically during the light phase. These data indicate that FLX exerts estrogen-like antiobesity and hypophagic actions by modifying circadian feeding patterns, and suggest that estrogen regulates circadian feeding rhythm via serotonergic neurons in the CNS.

Silibinin decreases hepatic glucose production through the activation of gut-brain-liver axis in diabetic rats.

Silibinin, a flavonolignan derived from milk thistle (Silybum marianum), has been revealed to have a beneficial effect on improving diabetes-impaired glycemic control. However, the underlying mechanism is still unclear. In the present study, to evaluate whether the gut-brain-liver axis, an important neural pathway for the control of hepatic glucose production, is involved in silibinin-regulated glucose homeostasis, the expression of glucagon-like peptide-1 receptor (GLP1R) in the duodenum, activation of neurons in the nucleus of the solitary tract (NTS), as well as glycogen accumulation and expression of gluconeogenic enzymes in the livers of diabetic SHRSP·Z-Leprfa/IzmDmcr (SP·ZF) rats with 4-week oral administration of silibinin (100 and 300 mg kg-1 day-1) were evaluated. Common hepatic branch vagotomy was further conducted in high-fat diet/streptozotocin (HFD/STZ)-induced diabetic SD rats to confirm the role of the gut-brain-liver axis in silibinin-improved glycemic control. The results revealed a significant inhibition of fasting blood glucose after SP·ZF rats were administrated with silibinin for 4 weeks. The expression of GLP1R in the duodenum and the activation of neurons in the NTS increased, while hepatic glucose production decreased on silibinin administration. However, the hypoglycemic effect of silibinin was reversed by common hepatic branch vagotomy in diabetic SD rats. Our study suggested that silibinin may be useful as a potential functional food ingredient against diabetes by triggering the gut-brain-liver axis.

Trans-cinnamaldehyde promotes nitric oxide release via the protein kinase-B /v-Akt murine thymoma viral oncogene -endothelial nitric oxide synthase pathway to alleviate hypertension in SHR.Cg-Leprcp/NDmcr rats.

OBJECTIVE: To evaluate whether endothelial dysfunction and hypertension are prevented by trans-cinnamaldehyde (tCA) through the activation of endothelial nitric oxide synthase (eNOS). METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and stimulated with tCA to determine cell viability using the methyl thiazolyl tetrazolium assay. The effect of tCA on nitric oxide (NO) production was determined by diaminofluorescein-dyes in the absence or presence of inhibitors of eNOS, AMPK, PKA, and AKT. The effect of tCA on blood pressure was determined by the tail-cuff method in obesity spontaneous hypertension (SHR. Cg-Leprcp/NDmcr) rats. The phosphorylation of eNOS and protein expression of the insulin-signaling pathway (InsR-IRS1-PI3K-AKT) were measured by western blot. RESULTS: tCA at concentrations less than 100 ¦ÌM did not affect cell viability in cultured HUVECs. Stimulation with tCA promoted NO release in a time-dependent manner compared with the control group. tCA-treated HUVECs also significantly increased AKT-Ser473 and eNOS- Ser1177 phosphorylation. In SHR-CP rats, treatment with tCA at a dose of 40 mg/kg/day for 6 weeks markedly reduced the systolic blood pressure and diastolic blood pressure, increased the phosphorylation of AKT and eNOS, and increased urinary nitric oxidation. CONCLUSION: tCA attenuated endothelial dysfunction and reduced blood pressure in SHR-CP rats. The underlying mechanisms may involve the increase in AKT and eNOS phosphorylation and the release of eNOS-derived NO.

S-equol Exerts Estradiol-Like Anorectic Action with Minimal Stimulation of Estrogen Receptor-α in Ovariectomized Rats.

Chronic estrogen replacement in ovariectomized rats attenuates food intake and enhances c-Fos expression in the suprachiasmatic nucleus (SCN), specifically during the light phase. S-equol, a metabolite of daidzein, has a strong affinity for estrogen receptor (ER)-ß and exerts estrogenic activity. The purpose of the present study was to elucidate whether S-equol exerts an estrogen-like anorectic effect by modifying the regulation of the circadian feeding rhythm in ovariectomized rats. Ovariectomized female Wistar rats were divided into an estradiol (E2)-replaced group and cholesterol (vehicle; Veh)-treated group. These animals were fed either a standard diet or an S-equol-containing diet for 13 days. Then, the brain, uterus, and pituitary gland were collected along with blood samples. In the rats fed the standard diet, E2 replacement attenuated food intake (P < 0.001) and enhanced c-Fos expression in the SCN (P < 0.01) during the light phase. Dietary S-equol supplementation reduced food intake (P < 0.01) and increased c-Fos expression in the SCN (P < 0.01) in the Veh-treated rats but not in the E2-replaced rats during the light phase. Dietary S-equol did not alter ER-α expression in the medial preoptic area or the arcuate nucleus, nor did dietary S-equol affect pituitary gland weight or endometrial epithelial layer thickness. By contrast, E2 replacement not only markedly decreased ER-α expression in these brain areas (P < 0.001) but also increased both the pituitary gland weight (P < 0.001) and the endometrial epithelial layer thickness (P < 0.001). Thus, dietary S-equol acts as an anorectic by modifying the diurnal feeding pattern in a manner similar to E2 in ovariectomized rats; however, the mechanism of action is not likely to be mediated by ER-α. The data suggest a possibility that dietary S-equol could be an alternative to hormone replacement therapy for the prevention of hyperphagia and obesity with a lower risk of adverse effects induced by ER-α stimulation.

Reduced Production of Hydrogen Sulfide and Sulfane Sulfur Due to Low Cystathionine ß-Synthase Levels in Brain Astrocytes of Stroke-Prone Spontaneously Hypertensive Rats.

Stroke-prone spontaneously hypertensive rats (SHRSP/Izm; SHRSP) develop severe hypertension and die of cerebral stroke. However, the genetic mechanisms underlying their stroke susceptibility have not been clarified yet. In this study, we used astrocytes from the newborn brain cortex of spontaneously hypertensive rats (SHR/Izm; SHR) and SHRSP to find the difference of genetic characteristics. Astrocytes are known to have functions of vasodilation and nutrient uptake for neurons in the brain. The continuous generation of hydrogen peroxide (H2O2) dose-dependently causes cell death in astrocytes, and SHRSP was more vulnerable than SHR. We found that the total thiols decreased in SHRSP astrocytes but the total glutathione (GSH) did not change. Hydrogen sulfide (H2S), which is known to protect cells through anti-oxidant and vasodilatory effects, is produced by cystathionine ß-synthase (CBS) in astrocytes. We found that H2S production was significantly decreased in SHRSP as compared to SHR. This was caused by the decreasing expression of mRNA, protein and enzyme activity of CBS in astrocytes. We also found that astrocyte cell death from oxidative stress could be prevented by GYY4137 H2S donor. H2S is also known to cause protein S-sulfhydration to modify enzyme activity. Sulfane sulfur in astrocytes was significantly lower in SHRSP and decreased by CBS inhibitor. We showed that astrocytes in SHRSP vulnerable to oxidative stress may be caused by reduction of H2S through lower expression and activity of CBS.

Effect of Angelica acutiloba Extract on Blood flow Regulation in Stroke-prone Spontaneously Hypertensive Rats.

Angelica acutiloba Kitagawa (Yamato Toki) is a herbal medicine known to exhibit various health effects. In this study, we used a rat model to examine the effects of two different Yamato Toki extracts on the blood flow response of the instep of the hind paw. Twelve-week- old male stroke-prone spontaneously hypertensive rats (SHRSP) were orally given 200 mg of a water extract of Yamato Toki (n=6), 200 mg of an ethanol extract of Yamato Toki (n = 6), 30 mg/kg of nicardipine (n = 6) as a positive control and saline (n = 6) as a negative control. Blood flow was monitored continuously for 0, 1, 3, 6 and 24 hours after treatment. Rats given 200 mg of the ethanol extract of Yamato Toki demonstrated significantly greater blood flow compared with control rats at I hour after treatment. An ethanol extract of Yamato Toki from Nara administrated orally can increase blood flow in SHRSP.

Effects of Berberine on Adipose Tissues and Kidney Function in 3T3-L1 Cells and Spontaneously Hypertensive Rats.

We aimed to investigate the effect of berberine on adipose tissues, as well as its effect on renal injury in 3T3-L1 cells and spontaneously hypertensive rats. 3T3-L1 cells were cultured and treated with berberine (5-20 pM) from days 3 to 8. Berberine added to the cultured medium could significantly down-regulate transcription factors, including CCAAT/enhancer binding protein ß, CCAAT/enhancer binding protein a, and peroxisome pro liferator-activated receptor y, and suppress peroxisome proliferator-activated receptor target genes, such as adipocyte fatty acid binding protein and fatty acid synthase, and inhibit 3T3-Ll fibroblast differentiation to adipocytes. Male spontaneously hypertensive rats received either 150 mg/day of berberine or saline orally for 8 weeks. Compared with the control, berberine-treated rats exhibited significant reductions in body weight gain (p < 0.05), as well as retroperitoneal and mesenteric adipose tissues (p < 0.05). Berberine-treated rats significantly decreased urinary albumin excretion, a marker of renal injury (p < 0.05). Long-term treatment with berberine decreased the adipose tissues weight and attenuated renal injury in spontaneously hypertensive rats. Based on these results, berberine has an important role in regulating adipose tissues. These results suggest the protective effect of berberine on metabolic syndrome related diseases, such as renal injury.

Involvement of orexin-A neurons but not melanin-concentrating hormone neurons in the short-term regulation of food intake in rats.

In order to elucidate the involvement of melanin-concentrating hormone (MCH) and orexin-A (ORX-A) neurons of the perifornical/lateral hypothalamic areas (PF/LH) in the regulation of food intake induced by acutely reduced glucose availability, we examined the food intake response and c-Fos expression in the MCH and ORX-A neurons in the PF/LH during 2-deoxy-D-glucose (2DG)-induced glucoprivation (400 mg/kg; i.v.) and systemic insulin-induced hypoglycemia (5 U/kg; s.c.) in male Wistar rats. The administration of both 2DG and insulin stimulated food intake and induced c-Fos expression in the ORX-A neurons corresponding to food intake, but not in the MCH neurons. These data indicate that ORX-A neurons, but not MCH neurons, play a role in the short-term regulation of food intake, and that the input signals for the neurons containing MCH and ORX-A are different, and these neurons play different roles in the regulation of feeding behavior.

Caffeic acid phenethyl ester suppresses oxidative stress in 3T3-L1 adipocytes.

The generation of oxidative stress, characterized by enhanced reactive oxygen species (ROS) formation, has been found in obesity. ROS production was increased during the differentiation of 3T3-L1 cells into adipocytes. We previously reported that caffeic acid phenethyl ester (CAPE) suppresses 3T3-L1 differentiation to adipocytes through the inhibition of peroxisome proliferator-activated receptor γ. In this study, the preventive effect of CAPE on oxidative stress in 3T3-L1 cells was observed. The results were as follows: (1) ROS production during 3T3-L1 cell differentiation to adipocytes was significantly (p < 0.05) suppressed by CAPE treatment in a concentration-dependent manner, (2) with CAPE treatment, the extracellular superoxide dismutase mRNA expression level significantly increased, but the NOX4 mRNA expression level did not change, and (3) CAPE treatment significantly increased superoxide dismutase (SOD) activity in 3T3-L1 cells. From these results, we suggest that the increased oxidative stress in 3T3-L1 differentiation to adipocytes is attenuated by CAPE treatment. This attenuation may be partly caused by increased SOD production.

Caffeic acid phenethyl ester suppresses the production of pro-inflammatory cytokines in hypertrophic adipocytes through lipopolysaccharide-stimulated macrophages.

Obesity is a condition in which excess body fat accumulates due to lipids producing adipocytes and an increased number of differentiated mature cells. Recently, new findings have shown that macrophages infiltrate into adipose tissues and produce various pro-inflammatory cytokines in obese subjects. The inflammatory changes induced by the cross-talk between adipocytes and macrophages are critical for the pathophysiology of obesity and thus of metabolic syndrome. Caffeic acid phenethyl ester (CAPE) is known to have many functions, including antibacterial, anticancer and anti-inflammatory properties, but there is no evidence of its effect on the inflammatory responses in hypertrophic adipocytes through stimulation by macrophages. We investigated the effect of CAPE on macrophages and hypertrophic adipocytes in this study. CAPE significantly suppressed the levels of lipopolysaccharide (LPS)-induced interleukin (IL)-1-beta, tumor necrosis factor (TNF)-alpha and monocyte chemoattractant protein (MCP)-1 from a macrophage cell line, RAW264.7. Supernatants of stimulated RAW264.7 macrophages drastically increased mRNA levels of pro-inflammatory cytokines such as IL-6, MCP-1 and TNF-alpha in 3T3-L1 hypertrophic adipocytes. CAPE also significantly and dose-dependently reduced the gene expression of these cytokines. Our findings indicate that CAPE has inhibitory effects on the production of pro-inflammatory cytokines from LPS-stimulated RAW264.7 macrophages. In addition, CAPE suppressed gene expressions of cytokines under inflammatory conditions of hypertrophic adipocytes, suggesting that it may have the potential to suppress inflammation by macrophage infiltration into adipose tissue in obese patients.

Caffeic acid phenethyl ester suppresses the production of adipocytokines, leptin, tumor necrosis factor -alpha and resistin, during differentiation to adipocytes in 3T3-L1 cells.

We previously reported that caffeic acid phenethyl ester (CAPE) suppresses 3T3-L1 differentiation to adipocytes through the inhibition of peroxisome proliferator-activated receptor (PPAR) gamma, CCAAT/enhancer-binding protein (C/EBP) alpha, fatty acid synthase (Fas) and adipocytes-specific fatty acid binding protein 2 (aP2) expressions (Juman et al., Biol. Pharm. Bull., 33, 1484-1488 (2010)). In the present study, we confirmed that CAPE had inhibitory effects on increased glycerol-3-phosphate dehydrogenase (GPDH) activity and an increased insulin receptor substrate 1 (IRS-1). Our data show that treatment with 50 µM CAPE significantly reduced the levels of leptin (p<0.05), resistin (p<0.05) and tumor necrosis factor (TNF)-alpha (p<0.05) which are known to aid adipocytokines production in adipocytes. In 3T3-L1 cells, treatment of CAPE decreased the triglyceride deposition similar to resveratrol, which is known to have an inhibitory effect on 3T3-L1 differentiation to adipocytes. In conclusion, we found that CAPE suppresses the production and secretion of adipocytokines from mature adipocytes in 3T3-L1 cells.

New chromone derivative terminalianone from African plant Terminalia brownii Fresen (Combretaceae) in Tanzania.

A new chromone derivative named terminalianone (1) was isolated from the African plant, Terminalia brownii Fresen (Combretaceae) in Tanzania. Its structure was determined to be 7-hydroxy-3-[6'-hydroxyphenyl-2'-oxo-ethyl]chromone by FAB-MS and NMR spectral data.

Caffeic acid phenethyl ester inhibits differentiation to adipocytes in 3T3-L1 mouse fibroblasts.

We investigated the inhibitory effect of caffeic acid phenethyl ester (CAPE) on the differentiation of 3T3-L1 mouse fibroblasts to adipocytes. 3T3-L1 cells were differentiated for adipocytes given high glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1 microM dexamethasone (DEX), 500 microM isobutylmethylxanthine (IBMX), and 5 microg/ml insulin for 7 days. After differentiation, cells were stained with Oil-Red-O to detect oil droplets in adipocytes. Additionally, the cells were lysed and measured for triglyceride contents. Total RNA was isolated from differentiated cells on day 0, 4 and 7. Then, RNA was analyzed using reverse transcription (RT)-polymerase chain reaction (PCR). CAPE dose-dependently suppressed oil droplet accumulation and reduced the droplet size. These findings showed that CAPE at concentrations of 25 to 50 microM could significantly inhibit triglyceride deposition (p<0.05). Treatment of 3T3-L1 with CAPE reduced the mRNA levels of peroxisome proliferator-activated receptor (PPAR) gamma and CCAAT/enhancer-binding protein (C/EBPalpha). Fatty acid synthase (Fas) and adipocyte-specific fatty acid binding protein (aP2) are known to be associated with lipid metabolism in adipocytes, and both Fas mRNA and aP2 mRNA were significantly suppressed by CAPE treatment. These findings suggested that CAPE suppresses 3T3-L1 differentiation to adipocytes through inhibition of PPARgamma, C/EBPalpha, Fas and aP2 expression.

Black and green tea polyphenols attenuate blood pressure increases in stroke-prone spontaneously hypertensive rats.

Oxidative stress was reported to be involved not only in cardiovascular diseases, but also in hypertension. Epidemiologic studies indicated that tea consumption slightly reduces blood pressure. We conducted two studies to determine whether black and green tea can lower blood pressure (BP) in stroke-prone spontaneously hypertensive rats (SHRSP). Male SHRSP (n=15) were allowed to recover for 2 wk after a transmitter for measuring BP was implanted in the peritoneal cavity. The rats were divided into three groups: the control group consumed tap water (30 mL/d); the black tea polyphenol group (BTP) consumed water containing 3.5 g/L thearubigins, 0.6 g/L theaflavins, 0.5 g/L flavonols and 0.4 g/L catechins; and the green tea polyphenol group (GTP) consumed water containing 3.5 g/L catechins, 0.5 g/L flavonols and 1 g/L polymetric flavonoids. The telemetry system was used to measure BP, which were recorded continuously every 5 min for 24 h. During the daytime, systolic and diastolic BP were significantly lower in the BTP and GTP groups than in the controls. Protein expressions of catalase and phosphorylated myosin light chain (MLC-p) were measured in the aorta by Western blotting. GTP significantly increased catalase expression, and BTP and GTP significantly decreased MLC-p expression in the aorta. These data demonstrate that both black and green tea polyphenols attenuate blood pressure increases through their antioxidant properties in SHRSP. Furthermore, because the amounts of polyphenols used in this experiment correspond to those in approximately 1 L of tea, the regular consumption of black and green tea may also provide some protection against hypertension in humans.

Serum lipid effects of a monounsaturated (palmitoleic) fatty acid-rich diet based on macadamia nuts in healthy, young Japanese women.

1. Recent studies have identified potential beneficial effects of eating nuts, most of which have substantial amounts of monounsaturated fatty acids (MUFA). Macadamia nuts consist of 75% fat by weight, 80% of which is MUFA (palmitoleic acid). 2. To examine variations in serum lipid levels in response to a high-MUFA diet based on macadamia nuts, 3 week interventions of macadamia nuts, coconuts and butter were determined in young, healthy Japanese female students. 3. After 3 weeks intervention, serum concentrations of total cholesterol and low-density lipoprotein-cholesterol were significantly decreased in the macadamia nut and coconut diets and bodyweight and body mass index were decreased in the group fed macadamia nuts, although there were no statistically significant changes in the group fed butter.

Antioxidant nutrients and hypoxia/ischemia brain injury in rodents.

Cerebral ischemia and recirculation cause delayed neuronal death in rodents, such as Mongolian gerbils and stroke-prone spontaneously hypertensive rats (SHRSP), which were used as an experimental stroke model. It was documented that an enhanced nitric oxide production, the occurrence of apoptosis, and an attenuated redox regulatory system contribute to the development of delayed neuronal death. Many studies have suggested the beneficial antioxidant effects of antioxidant nutrients such as vitamin E, green tea extract, ginkgo biloba extract, resveratrol and niacin in cerebral ischemia and recirculation brain injury. These results are important in light of an attenuation of the deleterious consequences of oxidative stress in ischemia and recirculation injury.

Effect of Undaria pinnatifida (Wakame) on the development of cerebrovascular diseases in stroke-prone spontaneously hypertensive rats.

1. We showed that a nutritional factor was able to attenuate the development of hypertension and its related diseases in stroke-prone spontaneously hypertensive rats (SHRSP). In the present study, the effect of Wakame, an edible brown seaweed, on the development of stroke was examined in SHRSP. 2. We studied the treatment with 5% (w/w in a diet) Wakame powder in salt-loaded (0.5% NaCl in drinking water) SHRSP. Salt-loaded animals treated with 5% cellulose or kaolin were used as controls. Wakame significantly delayed the development of stroke signs (P < 0.05) and significantly improved the survival rate of salt-loaded SHRSP (P < 0.05). There was no significant difference in the elevation of blood pressure among the three groups during the observation period. 3. We isolated fucoxanthin, a carotinoid, from Wakame powder and studied its preventive effect on ischaemic cultured neuronal cell death. Fucoxanthin significantly attenuated neuronal cell injury in hypoxia and re-oxygenation (P < 0.05). 4. Based on these results, we conclude that Wakame has a beneficial effect on cerebrovascular diseases in SHRSP, independent of hypertension. It is possible that fucoxanthin in Wakame may have a preventive effect against ischaemic neuronal cell death seen in SHRSP with stroke.