Objective: In this study, the neuroprotective effects of a water-soluble extract from culture medium of Ganoderma lucidum mycelia (WER) on oxidative stress-induced injury were examined using H₂O₂-treated PC12 cells. Additionally, we investigated both the acute and chronic effects of WER on brain necrosis and apoptosis induced by hypoxia/ischemia (H/I) followed by reoxygenation in mice.
Methods: Viability and apoptosis index of H₂O₂-treated PC12 cells were determined by 3,4,5-dimethylthiazol-2-yl (MTT) assay and TUNEL staining, respectively. H/I in mice was induced by unilateral ligation of carotid artery and exposure of 8%O₂ for 30 min. Twenty-four hours after H/I, neurological deficits, cerebral infarction volume, and apoptosis level were evaluated.
Results: WER–pretreated PC12 cells showed an increased viability evaluated by MTT assay compared to untreated cells. TUNEL staining indicated that WER induced a concentration-dependent decrease of the number of apoptotic cells. In the mouse model of H/I, acute (pre-H/I) treatment of WER (1 g/kg, p.o.) did not affect neurological deficits, total plasma oxidative stress, cerebral lipid peroxidation, and infarction volume assessed 24-h after reoxygenation. However, chronic treatment of WER (1 g/kg, p.o., for 7 days) significantly improved these parameters compared with distilled water-treated mice. Moreover, chronic treatment of WER decreased the levels of apoptosis in two brain areas, the sensori-motor cortex and the CA1 of the hippocampus, analyzed by TUNEL and cleaved caspase-3 immunostaining.
Conclusion: These results show that daily intake of WER relieves the cerebral ischemic injury, which may be attributed to decrease of oxidative stress.

Objective: Lentinus edodes (Shiitake) is a very popular mushroom in Asian cuisine. The water-soluble extract from culture medium of Lentinus edodes mycelia (LEM), which is commercially available as a nutritional supplement, is prepared by hot-water treatment from a solid medium composed of bagasse and defatted-rice bran overgrown for about 4 months with its mycelia. LEM was previously reported to have antioxidant activity and to suppress various oxidative damages. In this study, the neuroprotective effects of 2-week intake of LEM on cerebral ischemic damage induced by hypoxia/ischemia (H/I) followed by reoxygenation in mice were examined.
Method: Male C57BL/6J mice were divided into three groups, fed for two weeks with the control laboratory powder chow, 0.5% LEM-contained chow, or 1% LEM-contained chow, respectively. Cerebral ischemic damage was induced in the mice by H/I (i.e., unilateral ligation of the carotid artery and exposure of 8%O₂ for 30 min). Twenty-four hours after H/I, total plasma oxidative stress, neurological deficits, cerebral infarction volume were evaluated in each group. Furthermore, the number of apoptotic cells in ischemic penumbra, the hippocampal CA1 and CA2, and the somatosensory area of the cortex, were analyzed by TUNEL staining and cleaved caspase-3 immunostaining.
Results: The infarct area assessed 24-h after H/I was extended to the corpus striatum and cortex in the control mice. Treatment of LEM dose dependently improved plasma oxidative stress, neurological deficits, and cerebral infarction volume. Moreover, LEM decreased the levels of dihydroethidium activity as an index of super oxide production and the number of apoptotic cells in ischemic penumbra.
Conclusion: These results show that chronic intake of LEM relieves the hypoxia-induced cerebral ischemic injury, which may be attributed to the antioxidant effects of LEM.

Objective: Recently, we reported that long-term intake of a water-soluble extract from culture medium of Ganoderma lucidum mycelia (WER) reduced hyperglycemia and enhanced glucose transporter-4 (GLUT4) translocation to the plasma membrane in skeletal muscles and adipose tissue in KK-A^y mice, a type 2 diabetic animal model with obesity. In the present study, we investigated the effect of WER on hepatic carbohydrate metabolism.
Methods: Female KK-A^y mice were given free access to water and high-fat food containing 0.5% WER for 8 weeks, and blood glucose levels were assessed every week. At the end of the experimental period, the expression and activities of sugar metabolic enzymes in the liver were determined by Real Time RT-PCR and each activity measurement method. Also, the amount of glycogen was measured by anthrone-sulfuric acid method. Furthermore, the expression level of GLUT2 and activation of AMP kinase (AMPK) and glycogen synthase kinase 3β (GSk3β) was also determined by western blot analysis.
Results: The mice with the high-fat ingestion showed a gradual increase in the levels of blood glucose and body weight. In the WER-treated mice, the blood glucose level was suppressed after 2 weeks of intake. The gene expression and enzyme activities of both glucose-6-phosphatase and phosphoenolpyruvate carboxykinase were suppressed, whereas those of glucokinase were increased in the mice with WER intake and pioglitazone administration. The accumulation of glycogen was increased. Moreover the expression of GLUT2 and phosphorylation levels of AMPK and GSk3β were also increased in the mice with WER intake.
Conclusion: These results indicate that WER affects hepatic carbohydrate metabolism, which may derive from the suppression of gluconeogenesis through the modulation of related enzymes and enhancement of glucose uptake, glycolysis and glycogen synthesis.

Objective: Ganoderma lucidum (Rei-shi) is widely used as an alternative medicine agent to promote health and longevity. The water-soluble extract from culture medium of Ganoderma lucidum mycelia (WER) is prepared from a solid medium composed of bagasse and rice bran overgrown for 3–4 months with Ganoderma lucidum mycelia.
Design: In this study, we evaluate antioxidant activity of WER in vitro, and examined the effects of oral treatment of WER on oxidative stress in streptozotocin (STZ)-induced diabetic mice.
Method: Male ICR mice were treated with STZ (150 mg/kg, i.p.) and were housed for a week for induction of experimental diabetic state. WER (1 g/kg daily) was orally administered for an additional 9 weeks, and the levels of blood glucose, oxidative stress in plasma, liver and kidney of the mice were assessed.
Result: WER inhibited generation of superoxide anion and lipid peroxidation in concentration dependent manner in vitro. Oral administration of WER to diabetic mice significantly reduced the levels of blood glucose, hydroperoxides, triglyceride, ALT, and AST. Moreover, WER prevented the increase in lipid peroxide levels and the decrease in activity of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase) in liver and kidney of diabetic mice. In addition, histochemical studies revealed that treatment of WER precluded the sinusoidal dilation in liver and expansion of mesangial matrix in kidney of diabetic mice. Normal mice treated with WER showed no change in any parameters studied.
Conclusion: These data suggested that WER can act as an antioxidant in vivo, and show the antidiabetic effects by relieving diabetic hyperglycemia-induced oxidative stress.

Objective: The water-soluble extract of Ganoderma lucidum mycelia (WER) is prepared from a solid medium composed of bagasse and rice bran overgrown with Ganoderma lucidum mycelia. Recently, we reported that WER shows a blood glucose-lowering effect in maltose-loaded non-diabetic mice. Here, we investigated the efficacy of WER in type 2 diabetic state using KK-A^y mice. Moreover, the food-drug interactions of WER with α-glucosidase inhibitors, voglibose or acarbose were examined using both in vitro and in vivo experiments.
Methods: The glucose-lowering effects of oral administration in vivo of WER alone, or concomitant administration of WER with voglibose/acarbose on the elevation of blood glucose levels by sugar-tolerance tests were examined in KK-A^y mice. The inhibitory effects on α-glucosidase in vitro were also evaluated.
Results: Oral administration of WER (1 g/kg), which did not affect fasting blood glucose, significantly suppressed the hyperglycemia after loading of maltose (18% of decrease in AUC) compared to the water-administrated control mice. In vitro study showed that WER inhibited maltase in concentration-dependent manner. The inhibitory effects of lower concentrations of voglibose or acarbose on α-glucosidase activity were additively enhanced by the presence of WER, but those of higher concentrations were not affected. The glucose-lowering effect of voglibose (0.1 mg/kg) disappeared in maltose-loaded KK-A^y mice when the drug was concomitantly administrated with WER (1 g/kg), whereas acarbose (16 mg/kg) with WER showed no significant change in its effect.
Conclusion: These results demonstrated that WER shows the glucose-lowering effect in maltose-loaded KK-A^y, which may be based on inhibition of the α-glucosidase activity. The present study suggests that concomitant intake of WER with voglibose may override the therapeutic effect of voglibose on postprandial hyperglycemia by food-drug interaction in diabetic state.

Objective: The water-soluble extract of Ganoderma lucidum mycelia (WER), which is used as a health food, reduced hyperglycemia and enhanced glucose transporter 4 (GLUT4) translocation to the plasma membrane in skeletal muscle and adipose tissue in KK-A^y mice, a type 2 diabetic animal model with obesity.In order to elucidate the reduction of hyperglycemia by WER, we investigated the translocation of glucose transporter 4, glucose uptake and associating signal transduction in rat skeletal muscle (L6) cells. Method: The glucose uptake was analyzed with radioactive 2-deoxy-D-glucose.The localization of GLUT4 in L6 cells treated with various concentrations of WER was analyzed with immunohistochemical staining and Western blot technique.As a positive control, insulin or troglitazone was used in these experiments.Furthermore, the activation of intracellular signaling pathways by Western blot analysis and the influence of glucose uptake using four kinds of inhibitors (LY294002 as potent PI3K inhibitor, rapamycin as mTOR inhibitor, Gö6983 as broad PKC inhibitor, compound C as AMPK inhibitor) was evaluated. Results: GLUT4 protein content in the plasma membrane was induced in a dose-dependent manner of WER without increasing the gene expression and amount of total protein in the L6 cells and the glucose uptake was augmented with increasing the amount of GLUT4 translocated on the plasma membrane.The phosphorylation of phosphatidylinositol-3 kinase (PI3K), Akt and acetyl CoA carboxylase (ACC) were induced in a concentration dependent manner and inhibited by the above inhibitors except rapamycin. Conclusion: These results indicate that the hypoglycemic effect of some material(s) in WER may be due to the enhancement of glucose uptake through GLUT4 translocation on the plasma membrane by activating the PI3K/Akt pathway through improving insulin resistance.

Objective: The water-soluble extract of Ganoderma lucidum mycelia (WER) is prepared from a solid medium composed of bagasse and rice bran overgrown with Ganoderma lucidum mycelia. Recently, we have reported that WER had glucose-lowering effect in streptozotosin-induced diabetic mice, an animal model of type 1 diabetes. Here, we investigated whether long-term treatment with WER affects hyperglycemia and insulin resistance in KK-A^y mice, a type 2 diabetic animal model with obesity.
Methods: Female KK-A^y mice were given free access to water and high-fat food containing 0.5% WER for 8 weeks, with blood glucose and plasma insulin levels assessed every week. At the end of the experimental period, insulin tolerance test (ITT) was performed, and plasma levels of triglyceride, total cholesterol, HDL-cholesterol, AST, ALT and adiponectin were measured. Furthermore, expression of GLUT4 in skeletal muscle cell membrane and adipocytes was also determined by immunostaining and Western blot analysis.
Results: The mice with high-fat ingestion showed a gradual increase in levels of blood glucose and body weight. In the WER-treated mice, the blood glucose level was significantly suppressed after 2 weeks of treatment. WER also reduced plasma levels of ALT and insulin, but did not affect the other parameters. Additionally, ITT revealed that WER improved insulin sensitivity. Moreover, expression of GLUT4 in the plasma membrane of skeletal muscle cells and adipocytes of the WER-treated mice was increased.
Conclusion: These results indicate that WER has a glucose-lowering effect in type 2 diabetic mice. WER also improved hyperinsulinemia and insulin sensitivity, which may derive from enhancement of glucose uptake through GLUT4 of skeletal muscle cells and adipocytes.