Ganoderma lucidum extract stimulates glucose uptake in L 6 rat skeletal muscle cells

1Department of Pharmacology, 2Department of Biochemistry, College of Medicine, Kyung Hee University, Seoul; 3Department of Obesity Science, Dongduk Women’s University, Seoul; 4Department of Food and Biotechnology, Hanseo University, Seosan; 5Division of Rheumatology, Department of Internal Medicine, Kyung Hee Medical Center, Seoul, Republic of Korea; 1⁄2e-mail: 94princess@hanmail.net, ysvin@khu.ac.kr


INTRODUCTION
Ganoderma lucidum is an important medicinal fungus belonging to the Ganodermataceae family which has been popularly used for its health promoting properties (Stapleton et al., 1996;Vavvas et al., 1997;Thornton et al., 1998;Wasser & Weis, 1999).It is consumed more for its pharmaceutical rather than nutritional value.The effects of G. lucidum extract on cancer, hypertension, hypercholesterolemia, and hepatitis have been demonstrated by several studies (Franz, 1989;Furusawa et al., 1992;Wang et al., 1997).In addition, it was reported that G. lucidum extract showed hypoglycemic activity by increasing plasma insulin and by affecting hepatic enzymes in alloxan-induced diabetic mice (Hikino et al., 1985).Zhang & Lin, (2004) reported that G. lucidum extract showed protective effect on alloxan-induced pancreatic islet damage.
Given that skeletal muscles account for more than 80% of insulin-stimulated glucose uptake, an impaired glucose uptake in skeletal muscle is responsible for the development of type II diabetes mellitus (Baron et al., 1991).Two major regulatory molecules of glucose uptake, phosphatidylinositol (PI) 3-kinase and AMP-activated protein kinase (AMPK), are responsible for glucose uptake into the cytoplasm (Musi et al., 2001;Xi et al., 2001).PI 3-kinase is stimulated by insulin and mediates glucose uptake (Xi et al., 2001).Activation of PI 3-kinase in impaired skeletal muscle showed a reduced effect on glucose uptake (Bjornholm et al., 1997;Dohm et al., 1998).Protein kinase B (PKB) is an enzyme that can be activated by insulin via a pathway involving a PI 3-kinase dependent mechanism and can promote glucose uptake (Chan et al., 1999).AMPK mediates contraction-induced glucose uptake AMPK α, the catalytic subunits of AMPK, exists as two isoforms, α1 and α2.AMPK α1 is ubiquitously expressed in various tissues such as skeletal muscle, liver, and pancreas, while AMPK α2 is more limitedly expressed in skeletal and cardiac muscles (Cheung et al., 2000;Musi & Goodyear, 2002).
Although a hypoglycemic effect of G. lucidum extract has been reported, the exact mechanism of this effect has yet to be elucidated.Therefore, we investigated the effect of G. lucidum extract on glucose uptake in skeletal muscle cells by assessing the activities of PI 3-kinase and AMPK, two crucial mediators in the glucose uptake pathway.

MATERIALS AND METHODS
Preparation of extract.G. lucidum was purchased at Kyungdong market (Seoul, Korea) and was authenticated by the College of Oriental Medicine, Semyung University.Methanol extract of G. lucidum (yield: 19.7% of dry weight) was obtained by 48 h maceration at room temperature and was filtered through a 0.45 µm filter (Osmonics, Minnetonka, MN, USA), lyophilized, and kept at 4°C.
Cell culture.Monolayer of L6 skeletal muscle cells was maintained at subconfluent conditions in growth media containing DMEM with 0.045 g/ml glucose, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum.Cells were maintained in a humidified 37°C incubator with ambient oxygen and 5% CO 2 .
Glucose uptake assay.Glucose uptake was determined as previously described (Eichhorn et al., 2001).Cells were cultured on 12-well culture plates, washed with Krebs-Ringer phosphate buffer (KRB) (pH 7.4, 25 mM Hepes, 118 mM NaCl, 4.8 mM KCl, 1.3 mM CaCl 2 , 1.2 mM KH 2 PO 4 , 1.3 mM MgSO 4 , 5 mM NaHCO 3 ), 0.07% bovine serum albumin, and 5.5 mM glucose and incubated in KRB for 60 min.Cells were treated with 100 nM insulin, or 20, 50, or 100 µg/ml of G. lucidum extract for 30 min.Glucose uptake was measured by adding 20 µl glucose mixture (5 mM 2-deoxyglucose and 0.5 µCi 2-[ 3 H]deoxy-d-glucose in KRB) to 980 µl KRB followed by incubation for 20 min at 37°C.Nonspecific glucose uptake was measured by parallel incubations in the presence of 10 µM cytochalasin B, which blocks transporter-mediated glucose uptake, and was subtracted from the total uptake in each assay.Uptake was terminated by washing the cells three times with 1 ml ice-cold phosphate-buffered saline (PBS).Cells were subsequently lysed with 0.5 ml of 0.5 M NaOH solution containing 0.1% SDS, and the solution was rotated for 15 min.Cell associated radioactivity was measured in a liquid scintillation counter (PerkinElmer Life and Analytical Sciences).
Immunoblotting.The cell lysates were centrifuged for 15 min at 12 000 × g at 4°C, and the supernatant was collected.Proteins were separated by SDS/PAGE and transferred onto nitrocellulose membrane (Schleicher & Schuell, Middlesex, UK).After transfer, the membrane was blocked in 5% nonfat milk in Tris-buffered saline plus 0.1% Tween 20 (TBS-T) and then was incubated for 1 h at room temperature with 1 : 1000 diluted antibody against phospho-AMPK α1, α2, p38 MAPK or β-actin (Cell Signaling Technology, Beverly, MA, USA).Next, the membrane was washed in TBS-T followed by incubation with a horseradish peroxidase-conjugated secondary antibody (1:2000).The immunoreactive bands were visualized with an enhanced chemiluminescence kit (Amersham Pharmacia, Uppsala, Sweden).
Statistical analysis.Statistical analysis was performed using Student's t-test and one way analysis of variance (one way-ANOVA).The accepted level of significance was preset as P value < 0.05.Data are presented as means ± S.E.M.All calculations were performed using SPSS software (SPSS, Inc., Chicago, IL, USA).

G. lucidum extract stimulates glucose uptake in skeletal muscle cells
We determined the effect of G. lucidum extract on glucose uptake using 2-[ 3 H]deoxy-d-glucose in L6 skeletal muscle cells.Figure 1 shows that glucose uptake was increased by G. lucidum extract by about 85-90% compared to control (P < 0.01).The stimulatory effect of G. lucidum extract was dose-independent at dosages ranging from 20 to 100 µg/ml.At the doses of 20, 50, and 100 µg/ml of the uptake G. lucidum extract was 183.4 ± 8.1, 195.3 ± 6.8, and 187.7 ± 9.4%, respectively, compared to control.The stimulatory effect of G. lucidum extract on glucose uptake was as strong as that of 100 nM insulin (180.4 ± 8.6%) (Fig. 1), which was used as a positive control (Maleppillil et al., 2005).These results indicate that G. lucidum extract stimulates glucose uptake in L6 skeletal muscle cells.Since the extract showed a dose-independent effect on glucose transport, the following experiments were set at 50 µg/ml G. lucidum extract.

G. lucidum extract-stimulated glucose uptake is mediated by PI 3-Kinase
PI 3-kinase plays a critical role in insulin-stimulated glucose uptake.We performed PI 3-kinase assay using [γ-32 P]ATP.In Fig. 2A, autoradiogram of a TLC plate shows the incorporation of 32 P into the 3' position of PI.PI-3-phosphate (PI-3-P), which is formed by PI 3-kinase activity, was markedly increased by 100 nM insulin for 30 min (about 6-fold compared to control).Insulin was used as a positive control (Ceddia et al., 2005).The levels of PI-3-P were increased by G. lucidum extract at 10, 30 and 60 min compared to that of control.Densitometry analysis revealed that G. lucidum extract-stimulated PI 3-kinase activities at 10, 30, and 60 min were 5.8, 6.7, and 7.3-fold higher compared to those of the control, respectively.The G. lucidum extract-stimulated PI 3-kinase activity was attenuated by more than 90% by LY294002.The increase of PI 3-kinase activity by G. lucidum extract was significantly greater than that by 100 nM insulin (P < 0.05).As shown in Fig. 2B, the phosphorylation level of PKB, a downstream effecter of PI 3-kinase, was increased by treatment with G. lucidum extract in a time-dependent manner.Treatment with LY294002 almost completely inhibited the G. lucidum extractstimulated phosphorylation level of PKB.

G. lucidum extract-stimulated glucose uptake is mediated by AMPK, and p38 MAPK
To determine if the G. lucidum extract-stimulated glucose uptake was mediated by AMPK, we examined the phosphorylation levels of AMPK α1, α2 (catalytic subunits of AMPK) at Thr172 and p38 mitogen activated protein-kinase (p38 MAPK) proteins.5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR), a compound that activates AMPK, was then used as a positive control (500 µM) (Salt et al., 2000).As shown in Fig. 3A, the phosphorylation level of AMPK α1 was clearly increased by G. lucidum extract at 10, 30 and 60 min.The increased phosphorylation of AMPK α1 by G. lucidum extract was even greater than that by AICAR.Not as strong as for AMPK α1, a time-dependent increase in the phosphorylation level of AMPK α2 was also observed at 10, 30 and 60 min.p38 MAPK phosphorylation was accordingly increased at 10, 30 and 60 min (Fig. 3A).In an additional experiment, to determine whether AMPK and PI 3-kinase are component of the same signaling route, we investigated the phosphorylation level expression of PKB after treatment with AICAR in L6 skeletal muscle cells.The phosphorylation level of PKB was increased by insulin but not by AICAR (Fig. 3B).This result suggests that AMPK and PI 3-kinase may be component of different signaling route in L6 skeletal muscle cells.The intensity of β-actin, which was used as an internal control, was content.

DISCUSSION
G. lucidum extract has been used for long time in China to prevent and treat various human diseases such as hepatitis, hypertension, and hyperglycemic, turmorigenic and immunological diseases (Kabir et al., 1988;Kim et al., 1999;Cao et al., 2005;Kimura, 2005).Although a hypoglycemic effect of G. lucidum extract has been reported, its mechanism is not fully understood.
In skeletal muscle, glucose transport can be activated by at least two regulatory molecules, PI 3-kinase which is stimulated by insulin, and AMPK which is induced through muscle contraction (Zierath et al., 2000).PI 3-kinase activity has been suggested to be required for insulin-induced glucose uptake in skeletal muscle (Cheatham et al., 1994) and PKB, a downstream mediator beyond PI 3-kinase,  has been also implicated in insulin-stimulated glucose uptake (Brazil et al., 2004).To identify the signaling regulatory molecules involved in G. lucidum extract-stimulated glucose uptake, we investigated PI 3-kinase and PKB activities.As shown in Fig. 2A, G. lucidum extract stimulated PI 3-kinase with increasing time (10, 30, and 60 min) and this-stimulated PI 3-kinase activity was decreased to basal line by LY294002.Moreover, PKB, a downstream mediator of PI 3-kinase, was also increased by G. lucidum extract.These data showing that PI 3-kinase is activated by G. lucidum extract treatment suggest that PI 3-kinase is involved in G. lucidum extract-stimulated glucose uptake in L6 skeletal muscle cells.
AMPK is another regulatory protein in the glucose uptake pathway and energy metabolism (Cortright & Dohm, 1997;Brozinick & Birnbaum, 1998).Activation of AMPK leads to the activation of p38 MAPK, implicating p38 MAPK as a downstream effecter of AMPK (Xi et al., 2001) We found that treatment of skeletal muscle cells with G. lucidum extract increased the phosphorylation levels of AMPK α1, α2 at Thr-172, and of p38 MAPK (Fig. 3A).Ha et al. (2006) reported that topiramate stimulated the rate of glucose transport in L6 skeletal muscle cells.From these results, we suggest that G. lucidum extract also increases glucose uptake by stimulating AMPK activity in skeletal muscle cells.
Recently, several reports have suggested that AMPK and PI 3-kinase are elements of one signaling route and cross talk to each other (Ouchi et al., 2004;Zou et al., 2004;Longnus et al., 2005) Others, however, considered AMPK and PI 3-kinase to be on separate signaling pathways in skeletal muscle (Hayashi et al., 1998;Zierath et al., 2000;Mu et al., 2001).In our experiments, it was found that PI 3-kinase and AMPK might be on different routes in mediating glucose uptake in L6 skeletal muscle cells (Fig. 3B).
In conclusion, we found that G. lucidum extract significantly stimulated glucose uptake in L6 skeletal muscle cells.An interesting result was that the increase of glucose uptake by G. lucidum extract might be mediated by both PI 3-kinase and AMPK.

Figure 1 .
Figure 1.G. lucidum extract stimulates the rate of 2-[ 3 H]deoxy-d-glucose uptake in L6 skeletal muscle cells.Cells were treated with various doses of G. lucidum extract (20-100 µg/ml) for 30 min and 2-[ 3 H]deoxy-d-glucose uptake was measured in a scintillation counter.Insulin (100 nM) was used as a positive control.Data represent the mean ± standard error of five separate experiments, each performed in triplicate and presented as % control.Ins, insulin; GL, G. lucidum extract, *P < 0.05 vs. control.

Figure 2 .
Figure 2. Effect of G. lucidum extract on phosphatidylinositol 3-kinase (PI 3-kinase) activity and phosphorylaton of protein kinase B (PKB).PI 3-kinase activity was measured in anti-phosphotyrosine immunocomplex.Cells were treated with 50 µg/ml G. lucidum extract and insulin (100 nM, for 30 min) was used as a positive control (A).The phosphorylation of PKB was analyzed by immunoblotting with antibodies specific to phosphorylation of PKB.Cells were treated with 50 µg/ml G. lucidum extract in the absence or presence of 20 µM LY294002 (PI 3-kinase inhibitor) and 100 nM insulin for the indicated time.Beta actin was used as an internal control (B).Con, control; Ins, insulin; GL, G. lucidum extract; PI-3-P, phosphatidylinositol-3-phosphate; LY, LY294002; p-PKB, phospho-protein kinase B. *P < 0.05 vs. control.