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Morus alba leaf extract stimulates 5′-AMP-activated protein kinase in isolated rat skeletal muscle
Journal of Ethnopharmacology 122 (2009) 54–59
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Morus alba leaf extract stimulates 5 -AMP-activated protein kinase in isolated rat skeletal muscle Xiao Ma a , Nobumasa Iwanaka a , Shinya Masuda a , Kouhei Karaike a , Tatsuro Egawa a , Taku Hamada b , Taro Toyoda c , Licht Miyamoto d , Kazuwa Nakao d , Tatsuya Hayashi a,∗ a
Kyoto University Graduate School of Human and Environmental Studies, Yoshida-Nihonmatsu-Cho, Sakyo-ku, Kyoto 606-8501, Japan Graduate School of Sport and Exercise Science, Osaka University of Health and Sport Sciences, Osaka 590-0496, Japan Division of Food Science and Biotechnology, Kyoto University Graduate School of Agriculture, Kyoto 606-8502, Japan d Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan b c
a r t i c l e
i n f o
Article history: Received 29 July 2008 Received in revised form 18 November 2008 Accepted 25 November 2008 Available online 3 December 2008 Keywords: Morus alba 5 -AMP-activated protein kinase Diabetes mellitus Glucose transport Skeletal muscle Insulin
a b s t r a c t Aim of the study: Morus alba (mulberry) leaf is a natural therapeutic agent that has been shown to have an antidiabetic effect. We explored the possibility that 5 -AMP-activated protein kinase (AMPK) is involved in metabolic enhancement by the Morus alba leaf. Materials and methods: Isolated rat epitrochlearis muscle was incubated in a buffer containing Morus alba leaf hot water extract (MLE) and the AMPK activation and related events were examined. Results: In response to MLE treatment, the Thr172 phosphorylation of the catalytic ␣ subunit of AMPK, an essential step for full kinase activation increased in a dose- and time-dependent manner. Ser79 phosphorylation of acetyl CoA carboxylase, an intracellular substrate of AMPK, increased similarly. Analysis of isoform-specific AMPK activity revealed that MLE activated both the ␣1 and ␣2 isoforms of the catalytic subunit. This increase in enzyme activity was associated with an increased rate of 3-O-methyl-d-glucose transport in the absence of insulin and with phosphorylation of AS160, a signaling intermediary leading to glucose transporter 4 translocation. The intracellular energy status, estimated from the ATP and phosphocreatine concentrations, was not affected by MLE. Conclusion: MLE stimulates skeletal muscle AMPK activity acutely without changing the intracellular energy status. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Morus alba (mulberry; family: Moraceae) is cultivated in East Asian countries, such as China, Japan, and Korea. Its leaf is mainly used in feeding silkworms (Bombyx mori L.), although it has also been used in Chinese herbal medicine for centuries. Morus alba leaf is believed to contain substances that are beneficial in preventing and alleviating “Xiao Ke” (diabetes) and is consumed as a healthpromoting drink, mulberry tea. A recent study showed that coingestion of Morus alba extract with 75 g of sucrose significantly attenuated the increase in blood glucose concentration observed over the initial 120 min of testing in nondiabetic and Type 2 diabetic individuals (Mudra et al., 2007). Morus alba leaf contains active compounds that can inhibit ␣-galactosidases, such as 1-deoxynojirimycin (Asano et al., 2001; Miyahara et al., 2004), and this effect may help
∗ Corresponding author. Tel.: +81 75 7536640; fax: +81 75 7536640. E-mail address: [email protected] (T. Hayashi). 0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.11.022
suppress postprandial hyperglycemia by reducing the rate of digestion and absorption of carbohydrates from intestines. However, intraperitoneal administration of Morus alba leaf extract has a hypoglycemic effect in streptozotocin-induced hypoinsulinemic diabetic mice (Chen et al., 1995; Kimura et al., 1995). Morus alba leaf extract also increases the rate of glucose uptake in isolated mouse diaphragm in the absence of insulin (Chen et al., 1995). These observations suggest that the acute hypoglycemic effect is caused by a direct enhancement of insulin-independent glucose transport in skeletal muscle, the major site of whole-body glucose uptake and utilization (Defronzo, 1988; Goodyear and Kahn, 1998). We hypothesized that 5 -AMP-activated protein kinase (AMPK) is involved in the antidiabetic effect of Morus alba leaf. AMPK is the major signaling intermediary in the exercise-stimulated insulinindependent glucose transport in skeletal muscle (reviewed in Kahn et al., 2005; Hardie et al., 2006; Musi and Goodyear, 2006). AMPK is a heterotrimeric kinase comprising a catalytic ␣ subunit and two regulatory subunits,  and ␥. There are two distinct ␣ isoforms in skeletal muscle: AMPK␣1 and AMPK␣2 (Stapleton et
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al., 1996), and both isoforms are activated in response to muscle contraction, which stimulates glucose transport in the absence of insulin (Hayashi et al., 1998, 2000; Mu et al., 2001; Toyoda et al., 2006). Skeletal muscle AMPK is also implicated in a variety of antidiabetic properties of exercise, including glucose transporter 4 (GLUT4) expression (Zheng et al., 2001; Holmes et al., 2004; Nakano et al., 2006), glycogen regulation (Wojtaszewski et al., 2002; Jorgensen et al., 2004; Miyamoto et al., 2006), fatty acid oxidation (Winder and Hardie, 1996; Hutber et al., 1997; Vavvas et al., 1997), and enhanced insulin sensitivity (Fiedler et al., 2001; Buhl et al., 2002; Iglesias et al., 2002; Pold et al., 2005; Nakano et al., 2006; Tanaka et al., 2007). Skeletal muscle AMPK mediates part of glucose and lipid homeostasis by adipokines, including leptin and adiponectin, and the hypoglycemic effect of metformin (reviewed in Kahn et al., 2005; Hardie et al., 2006; Musi and Goodyear, 2006). The purpose of this study was to clarify whether Morus alba leaf acts directly on skeletal muscle to stimulate AMPK. We evaluated the effect of Morus alba leaf hot water extract (MLE) on AMPK activity in isolated rat skeletal muscle incubated in vitro. 2. Materials and methods 2.1. Experimental animals Male Wistar rats, aged 5 weeks and weighing 100–120 g, were obtained from Shimizu Breeding Laboratories (Japan). The animals were housed in an animal room maintained at 23 ◦ C with a 12:12-h light–dark cycle and fed a standard laboratory diet and water ad libitum. All protocols for animal use and euthanasia were reviewed and approved by the Kyoto University Graduate School of Human and Environmental Studies, Kyoto University Graduate School of Medicine, and Kyoto University Radioisotope Research Center in Japan. The rats were randomly assigned to the experimental groups. 2.2. Hot water extraction of Morus alba leaf Dried Morus alba leaves (product number: JAN4976015300427, manufacturing date: February 2005) were purchased from the Nakamura Chiro Association (Japan). The leaves (4 g) were powered and extracted with 50 times (w/v) of hot water (85 ◦ C) for 3 h. The extract was filtered through filter paper (No. C030633, Toyo Roshi, Japan), concentrated to a volume of 1/20 (v/v) of the initial solution volume by heating at a nonboiling temperature near to 100 ◦ C, and then dried completely under vacuum at 25 ◦ C. The dried extract (w/w = 0.59 g, yield = 15%) was used as the MLE. 2.3. Muscle incubation Rat epitrochlearis muscle was treated as described previously (Hayashi et al., 1998; Toyoda et al., 2004, 2006) with modifications. The rats were killed by cervical dislocation, and the muscles were removed rapidly. Both ends of each muscle were tied with sutures, and the muscle was mounted on an incubation apparatus with the resting tension set to 0.5 g. To recover AMPK activation during the isolating procedure, the muscle was preincubated in 7 ml of Krebs–Ringer bicarbonate buffer (KRB): 117 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2 , 1.2 mM KH2 PO4 , 1.2 mM MgSO4 , and 24.6 mM NaHCO3 containing 2 mM of pyruvate (KRBP) for 60 min. The muscle was then incubated in 7 ml of fresh buffer containing various concentrations (0–14.3 mg/ml) of MLE. The muscles were also incubated with 2 mM of 5-aminoimidazole-4-carboxamide-1-d-ribonucleoside (AICAR) for 30 min, 500 M 2,4-dinitrophenol (DNP) for 15 min or 1 M insulin for 30 min. Our previous studies have shown that these protocols elicit maximal activation of AMPK (AICAR, DNP) or glucose transport (insulin) in isolated epitrochlearis muscle (Hayashi et al., 1998, 2000). To measure the
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time course of changes in AMPK phosphorylation, the muscle was incubated with MLE (4.28 mg/ml) for up to 60 min. The buffer was bubbled with gas continuously using a mixture of 95%O2 –5%CO2 and maintained at 37 ◦ C. The muscle was then either used fresh to measure glucose uptake (see 3-O-methyl-d-glucose transport), or frozen immediately in liquid nitrogen for the subsequent measurement of isoform-specific AMPK activity and adenosine triphosphate (ATP) and phosphocreatine (PCr) concentrations, and Western blot analysis. 2.4. Western blot analysis The muscle was homogenized in ice-cold lysis buffer (1:40 w/v) containing 20 mM of Tris–HCl (pH 7.4), 1% of Triton X, 50 mM of NaCl, 250 mM of sucrose, 50 mM of NaF, 5 mM of sodium pyrophosphate, 2 mM of dithiothreitol, 4 mg/l of leupeptin, 50 mg/l of trypsin inhibitor, 0.1 mM of benzamidine, and 0.5 mM of phenylmethylsulfonyl fluoride (Buffer A), and centrifuged at 16,060 × g for 30 min at 4 ◦ C. The supernatant (10 g of protein) was mixed with loading buffer and boiled, and the denatured proteins were separated on either a 10% polyacrylamide gel for AMPK and Akt, or a 6% gel for acetyl CoA carboxylase (ACC). The proteins were then transferred to polyvinylidene difluoride membranes at 100 V for 1 h. The membrane was blocked with Block Ace (Yukijirushi Nyugyo, Japan) overnight at 4 ◦ C, and then incubated with AMPK␣ (Cell Signaling Technology, USA), phosphospecific AMPK␣ Thr172 (Cell Signaling Technology, USA), phosphospecific ACC Ser79 (Upstate Biotechnology, USA), Akt (Cell Signaling Technology, USA), and phosphospecific Akt Ser473 (Cell Signaling Technology, USA). To immunoprecipitate the Akt substrate of the 160-kDa (AS160), an aliquot of supernatant was prepared as described above (300 g of protein) and incubated with anti-AS160 (Rab-GAP) (Upstate Biotechnology, USA) and protein A-Sepharose CL4B beads (Amersham Biosciences, Sweden) overnight with end-over-end rotation at 4 ◦ C. The immunoprecipitate was washed three times in Buffer A, and then centrifuged at 4000 × g for 30 s at 4 ◦ C. The supernatant was removed, and the beads were mixed with loading buffer and boiled. The denatured proteins were then separated on a 10% polyacrylamide gel. The membrane was blocked with Block Ace overnight at 4 ◦ C, and then incubated with anti-AS160 (Rab-GAP) or antiphospho-(Ser/Thr) Akt substrate (Cell Signaling Technology, USA) as described in by Arias et al. (2007). The membrane was washed, reacted with anti-rabbit IgG coupled to peroxidase, and developed with enhanced chemiluminescence reagents according to the manufacturer’s instructions (Amersham, UK). The signals on the blot were detected and quantified using a Lumino LAS-1000 System image analyzer (Fuji Photo Film, Japan). 2.5. Isoform-specific AMPK activity assay The AMPK activity was measured as described by Hayashi et al. (2000) and Toyoda et al. (2004) with modification. Muscle was homogenized in Buffer A, as described in the Western blot analysis subsection, and the resultant supernatant liquid (100 g of protein) was immunoprecipitated with isoform-specific antibodies directed against the ␣1 or ␣2 catalytic subunit of AMPK (Toyoda et al., 2004) and protein A-Sepharose CL4B beads. The immunoprecipitate was washed twice in both Buffer A and wash buffer (240 mM of HEPES, 480 mM of NaCl). The kinase reaction was performed in 40 mM of HEPES (pH 7.0), 0.1 mM of SAMS peptide (Hayashi et al., 2000; Toyoda et al., 2004), 0.2 mM of AMP, 80 mM of NaCl, 0.8 mM of dithiothreitol, 5 mM of MgCl2, 0.2 mM of ATP (2 Ci of [␥-32 P]ATP (NEN Life Science Products, USA), in a final volume of 40 l for 20 min at 30 ◦ C. At the end of the reaction, a 15 l aliquot was removed and spotted onto Whatman P81 paper (Whatman International, UK). The filter paper was washed at least six times in 1%
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phosphoric acid and once in acetone. 32 P incorporation was quantified using a scintillation counter, and kinase activity was expressed as pmoles of incorporated ATP per mg of immunoprecipitated protein per minute. 2.6. ATP and PCr assay The frozen muscle was homogenized in 0.2 M HClO4 (3:25 w/v) in an ethanol–dry ice bath (−20 to −30 ◦ C) and centrifuged at 16,060 × g for 2 min at −9 ◦ C. The supernatant of the homogenate was neutralized with a solution containing 2 M of KOH, 0.4 M of KCl, and 0.4 M of imidazole, centrifuged at 16,060 × g for 2 min at −9 ◦ C, and then subjected to enzymatic analysis (Hayashi et al., 2000). 2.7. 3-O-methyl-d-glucose transport The assay used to measure 3-O-methyl-d-glucose (3MG) transport was performed as described by Hayashi et al. (1998) and Toyoda et al. (2004) with modification. The muscle was transferred to 2 ml of KRB containing 1 mM of 3-O-[methyl-3 H]-d-glucose (1.5 Ci/ml) (American Radiolabeled Chemicals, USA) and 7 mM d[14 C]mannitol (0.3 Ci/ml) (NEN Life Science Products, USA) at 30 ◦ C and incubated for 10 min. The muscle was blotted onto filter paper, trimmed, frozen in liquid nitrogen, and stored at −80 ◦ C. The frozen muscle was weighed and incubated in 300 l of 1 M NaOH at 80 ◦ C for 10 min. The digestate was neutralized with 300 l of 1 M HCl and the particulates were precipitated by centrifugation at 20,000 × g for 2 min. The radioactivity in aliquots of the digested protein was measured using liquid scintillation counting for dual labels, and the extracellular and intracellular spaces were calculated. 2.8. Measurement of osmolarity The osmolarity of the buffers was measured using a multisample osmometer (Osmette III, Precision Systems Inc., USA). 2.9. Statistical analysis The data are expressed as the mean ± S.E. The mean values were compared using a one-way analysis of variance (ANOVA) followed by a post hoc comparison with Fisher’s protected least-significant difference method. Two mean values ware compared using the Student’s t-test. Differences between the groups were considered significant for P < 0.05.
Fig. 1. Morus alba leaf extract (MLE) increases the phosphorylation of AMPK␣ and acetyl CoA carboxylase (ACC) in skeletal muscle. Isolated epitrochlearis muscle was preincubated for 60 min, and then incubated in the absence (basal), or presence of MLE (4.28 mg/ml) or AICAR (2 mM) for 30 min. The tissue lysate was subjected to Western blot analysis with phosphospecific antibodies to: (A) phosphorylated AMPK␣, (B) total AMPK␣, and (C) phosphorylated ACC.
2002). The marked phosphorylation of ACC paralleled the increase in AMPK phosphorylation (Fig. 1C). Similarly, AICAR stimulation caused significant phosphorylation of ACC (Fig. 1C). To determine the dose and time dependency of the AMPK phosphorylation, muscles were stimulated with MLE for various times and at various concentrations. The time course study revealed that 4.28 mg/ml of MLE induced acute phosphorylation of AMPK after an incubation period as short as 15 min, and that the peak increase occurred after an incubation period of 30–45 min (Fig. 2). The dose–response study showed a significant phosphorylation of 1.6-fold at a concentration of 4.28 mg/ml of MLE, and a 1.9-fold phosphorylation at a concentration of 14.3 mg/ml of MLE (Fig. 3). 3.2. MLE activated both AMPK ˛1 and ˛2 in skeletal muscle To identify which catalytic subunit is activated by MLE, isoform-specific AMPK activity was measured in anti-␣1 and anti-␣2 immunoprecipitates of the epitrochlearis muscle after treatment with MLE. MLE stimulation significantly increased the activity of both AMPK␣1 and AMPK␣2: the activity of AMPK␣1 = 10.5 ± 3.2 pmol/mg/min in the basal condition and
3. Results 3.1. MLE increased phosphorylation of AMPK ˛ Thr172 and ACC Ser79 in skeletal muscle The Thr172 residue in both the ␣1 and ␣2 catalytic subunits is the primary site responsible for AMPK activation (Stein et al., 2000). To determine whether MLE activates AMPK, we measured the degree of phosphorylation of AMPK ␣Thr172 using Western blot analysis employing a phosphospecific antibody. Compared with the basal condition, muscles stimulated with MLE (4.28 mg/ml, 30 min) exhibited an increased phosphorylation of Thr172 without any change in the total amount of AMPK␣ (Fig. 1A and B). The AMPK activator AICAR (2 mM, 30 min) also caused a strong phosphorylation of AMPK (Fig. 1A and B). AICAR is taken up into skeletal muscle and metabolized by adenosine kinase to form ZMP, a monophosphorylated derivative that mimics the effects of AMP on AMPK (Hardie and Carling, 1997). ACC is a downstream target of AMPK in skeletal muscle, and the phosphorylation of the Ser79 site of ACC reflects the total AMPK activity (Davies et al., 1990; Park et al.,
Fig. 2. MLE increases phosphorylation of AMPK␣ in a time-dependent manner in skeletal muscle. Isolated epitrochlearis muscle was preincubated for 60 min, and then incubated in the presence of MLE (4.28 mg/ml) for 0, 15, 30, 45, or 60 min, or AICAR (2 mM) for 30 min. The tissue lysate was subjected to Western blot analysis with phosphospecific AMPK␣ antibody. The values are expressed as the mean ± S.E., n = 4 per group. *P < 0.05 and **P < 0.01 versus the 0 min incubation group.
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Fig. 3. MLE increases phosphorylation of AMPK␣ in a dose-dependent manner in skeletal muscle. Isolated epitrochlearis muscle was preincubated for 60 min, and then incubated in the presence of 0, 1.43, 4.28, or 14.30 mg/ml of MLE for 30 min, or AICAR (2 mM) for 30 min. The tissue lysate was subjected to Western blot analysis with phosphospecific AMPK␣ antibody. The values are expressed as the mean ± S.E., n = 6 (basal, MLE) and 4 (AICAR) per group. **P < 0.01 versus the basal condition (0 mg/ml).
Fig. 5. MLE increases 3-O-methyl-d-glucose transport (3MG) transport in skeletal muscle. Isolated epitrochlearis muscle was preincubated for 60 min, and then incubated in the absence (basal), or presence of MLE (4.28 mg/ml) or insulin (1 M) for 30 min, and the 3MG transport activity was determined. The values are expressed as the mean ± S.E., n = 7 (basal, MLE) and 4 (insulin) per group. **P < 0.01 versus the basal condition.
18.7 ± 2.9 pmol/mg/min after incubation with MLE; and the activity of AMPK2␣ = 8.9 ± 1.6 pmol/mg/min in the basal condition and 24.4 ± 1.7 pmol/mg/min after incubation with MLE (Fig. 4). We used DNP, a pharmacological inhibitor of oxidative phosphorylation, as a positive control for AMPK␣1 and ␣2 activation (Hayashi et al., 2000; Fig. 4).
Akt is a key molecule in the insulin-stimulated signaling pathway leading to enhanced glucose transport and metabolism (Ueki et al., 1998). Recent studies suggest that AS160 is a downstream nexus of both Akt-mediated and AMPK-mediated glucose transport in skeletal muscle (Kramer et al., 2006; Treebak et al., 2006), and that AS160 is an intracellular substrate of AMPK in skeletal muscle (Treebak et al., 2006). In our study, in parallel with the increased 3MG transport observed in the absence of insulin (Fig. 5), MLE stimulation did not change the phosphorylation status of Akt (Fig. 6A), but significantly increased phosphorylation of AS160, as observed by AICAR stimulation (Fig. 6B).
3.3. MLE stimulated glucose transport in skeletal muscle in the absence of insulin Next, we investigated whether the activation of AMPK in skeletal muscle by MLE is associated with enhanced glucose transport. The maximally effective dose of insulin (1 M, 30 min) increased the rate of 3MG transport by 5-fold compared with the basal condition (Fig. 5). In the absence of insulin, MLE stimulation (4.28 mg/dl, 30 min) increased the rate of 3MG transport by 1.4-fold (Fig. 5).
Fig. 4. MLE activates both AMPK␣1 and ␣2 activities in skeletal muscle. Isolated epitrochlearis muscle was preincubated for 60 min, and then incubated in the absence (basal), or presence of MLE (4.28 mg/ml) for 30 min or DNP (500 M) for 15 min. The isoform-specific AMPK activity was determined in the anti-AMPK␣1 and anti-AMPK␣2 immunoprecipitates. The values are expressed as the mean ± S.E., n = 7 (basal, MLE) and 4 (DNP) per group. *P < 0.05 and **P < 0.01 versus the basal condition.
3.4. MLE treatment did not affect ATP and PCr content in skeletal muscle AMPK is activated in response to energy-consuming stress, such as muscle contraction, hypoxia, and inhibition of oxidative phosphorylation (Hayashi et al., 2000). We measured the content of ATP and PCr to determine whether MLE increases AMPK activity by energy deprivation. MLE treatment (4.28 mg/ml, 30 min) had no effect on these indicators of muscle energy status (Table 1).
Fig. 6. MLE increases phosphorylation of AS160 but not of Akt. Isolated epitrochlearis muscle was preincubated for 60 min, and then incubated in the absence (basal), or presence of MLE (4.28 mg/ml), insulin (1 M) or AICAR (2 mM) for 30 min. Tissue lysates were subjected to Western blot analysis with phosphospecific or total Akt antibodies (A). Lysates were also subjected to immunoprecipitation with AS 160 (Rab-GAP) antibody, and then subjected to Western blot analysis with antiphospho-(Ser/Thr) Akt substrate or AS 160 (Rab-GAP) antibodies (B).
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Table 1 MLE does not change the ATP and PCr content in skeletal muscle.
Basal MLE
ATP (nmol/mg)
Phosphocreatine (nmol/mg)
4.4 ± 0.2 4.0 ± 0.3
16.6 ± 0.6 16.4 ± 0.4
Isolated epitrochlearis muscle was preincubated for 60 min, and then incubated in the absence (basal), or presence of MLE (4.28 mg/ml) for 30 min, and the intracellular ATP and PCr content measured. The values are expressed as mean ± S.E.; n = 5 per group.
Table 2 Addition of MLE to KRBP does not change the osmolarity. Osmolarity (mOms/kg) Saline KRBP MLE + KRBP
284.5 ± 0.5 274.3 ± 2.9 285.3 ± 0.6
The osmolarity of saline, KRBP, and KRBP containing MLE (4.28 mg/ml) was measured with an osmometer. The values are the mean ± S.E., n = 3 per group.
3.5. MLE treatment did not change the osmolarity of KRBP Incubating rat epitrochlearis muscle in hyperosmotic buffer (KRBP with 120 mM sorbitol) activates AMPK (Hayashi et al., 2000). The osmolarity of KRBP containing MLE (4.28 mg/ml) did not differ from that of saline or KRBP (Table 2). 4. Discussion The novel finding of our study is that incubation with MLE acutely increases AMPK phosphorylation (Figs. 1–3) and activity (Fig. 4), and correspondingly, increases insulin-independent 3MG transport activity (Fig. 5) in isolated rat skeletal muscle. Enhanced phosphorylation of ACC (Fig. 1C) and AS160 (Fig. 6B), both of which are downstream targets of AMPK, indicates an actual increase in kinase activity in vivo. The phosphospecific immunoblot for Thr172 (Figs. 1A, 2, and 3), an essential site for full kinase activity, indicates that MLE induces the covalent modification by an upstream kinase. We used the isolated muscle preparation incubated in vitro to eliminate the effect of systemic confounders, such as humoral and neuronal factors and blood flow. In particular, we considered the possibility that oral administration of MLE, which may contain ␣-galactosidase inhibitors (Asano et al., 2001; Miyahara et al., 2004), could evoke secondary metabolic changes in skeletal muscle. In addition, because the isolation procedure increases AMPK␣1 activity (Toyoda et al., 2006), we exposed muscles to MLE after a preincubation period (60 min) sufficient to stabilize AMPK␣1 activity to the basal level (Toyoda et al., 2006). Thus, our method made it possible to examine the direct effect of MLE on the activity of both AMPK␣1 and AMPK␣2. We chose rat epitrochlearis muscle because it is a flat, thin muscle capable of absorbing oxygen and nutrients from a buffer in vitro (Nesher et al., 1980a,b). Differential ATPase staining has demonstrated that this muscle is a mixture of both fast- and slow-twitch fibers, i.e., more than 80% of the fibers are fast-twitch fibers and 15% are slow-twitch fibers (Nesher et al., 1980a,b). We have not yet identified the active constituent of MLE. Recent reports have suggested that berberine, a natural isoquinoline alkaloid, stimulates glucose transport by activating AMPK in L6 cultured cells (Cheng et al., 2006; Lee et al., 2006). Berberine has been isolated in variety of medical plants, such as Coptis chinensis (family: Ranunculaceae), a Chinese herb that has been used to treat diabetes for thousands of years in Asia. Berberine seems to activate AMPK by decreasing intracellular energy production because it significantly increases the ratio of AMP:ATP (Cheng et al., 2006). In support of this idea, a more recent study has suggested that berberine, and its more
biologically available derivative dihydroberberine, inhibits cellular respiration by inhibiting complex 1 of the mitochondrial respiratory chain in L6 cells (Turner et al., 2008). These observations lead us to hypothesize that berberine may be the main active constituents of MLE. However, MLE stimulation did not reduce the concentration of either ATP or PCr (Table 1), and therefore, we concluded that it is unlikely that energy-depriving compounds such as berberine play a major role in MLE-induced AMPK activation in skeletal muscle. Although we did not examine the long-term effect of MLE on skeletal muscle or whole-body metabolism, studies have clearly shown that AICAR improves the metabolic profile in animals. In our earlier study, repeated activation of AMPK by intraperitoneal injection of AICAR, three times a day for 7 d, increased GLUT4 protein by 40%, and insulin-stimulated 2-deoxyglucose transport activity by 20% in mouse soleus muscle (Nakano et al., 2006). Similarly, AICAR injections over 7 d significantly improved the response to an oral glucose tolerance test and insulin tolerance test in KKAy -CETP mice, a model of insulin-resistant type 2 diabetes mellitus (Fiedler et al., 2001). In addition, subcutaneous injections of AICAR over 7 weeks normalized the response to an oral glucose tolerance test, and decreased fasting concentrations of glucose and insulin close to the level of lean controls in obese Zucker (fa/fa) rats (Buhl et al., 2002). In ZDF rats, which are characterized by a progressive cell dysfunction and a leptin receptor defect, AICAR treatment over a period of 8 weeks increased the whole-body insulin sensitivity, which was associated with the level of muscle GLUT4 expression, and preserved the -cell function (Pold et al., 2005). Taken together, these data lead us to speculate that repeated activation of AMPK by MLE may also achieve long-term metabolic benefits that could protect against the development of diabetes. In summary, for the first time, we have demonstrated that MLE increases the activity of both AMPK␣1 and ␣2 in skeletal muscle, and that this increase is associated with insulin-independent glucose transport without change in the energy status of the muscle. We propose that Morus alba leaf fosters a metabolic milieu that reduces the risk of type 2 diabetes, at least in part, by activating skeletal muscle AMPK. Acknowledgements We are grateful to Yuichi Kawano for technical assistance, and Kaoru Ijiri and Yoko Koyama for secretarial assistance. We also thank the Radioisotope Research Center of Kyoto University for instrumental support in the radioisotope experiments. Tatsuya Hayashi was supported by research grants from the Japan Society for the Promotion of Science (17500424 and 20500576). References Arias, E.B., Kim, J., Funai, K., Cartee, G.D., 2007. Prior exercise increases phosphorylation of Akt substrate of 160 kDa (AS160) in rat skeletal muscle. American Journal of Physiology—Endocrinology and Metabolism 292, E1191–E1200. Asano, N., Yamashita, T., Yasuda, K., Ikeda, K., Kizu, H., Kameda, Y., Kato, A., Nash, R.J., Lee, H.S., Ryu, K.S., 2001. Polyhydroxylated alkaloids isolated from mulberry trees (Morusalba L.) and silkworms (Bombyx mori L.). Journal of Agricultural and Food Chemistry 49, 4208–4213. Buhl, E.S., Jessen, N., Pold, R., Ledet, T., Flyvbjerg, A., Pedersen, S.B., Pedersen, O., Schmitz, O., Lund, S., 2002. Long-term AICAR administration reduces metabolic disturbances and lowers blood pressure in rats displaying features of the insulin resistance syndrome. Diabetes 51, 2199–2206. Chen, F., Nakashima, N., Kimura, I., Kimura, M., 1995. Hypoglycemic activity and mechanisms of extracts from mulberry leaves (folium mori) and cortex mori radicis in streptozotocin-induced diabetic mice. Yakugaku Zasshi 115, 476–482. Cheng, Z., Pang, T., Gu, M., Gao, A.H., Xie, C.M., Li, J.Y., Nan, F.J., Li, J., 2006. Berberinestimulated glucose uptake in L6 myotubes involves both AMPK and p38 MAPK. Biochimica et Biophysica Acta 1760, 1682–1689. Davies, S.P., Sim, A.T., Hardie, D.G., 1990. Location and function of three sites phosphorylated on rat acetyl-CoA carboxylase by the AMP-activated protein kinase. European Journal of Biochemistry 187, 183–190. Defronzo, R.A., 1988. Lilly lecture, 1987. The triumvirate: beta-cell, muscle, liver. A collusion responsible for NIDDM. Diabetes 37, 667–687.
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Morus alba leaf extract stimulates 5 -AMP-activated protein kinase in isolated rat skeletal muscle Xiao Ma a , Nobumasa Iwanaka a , Shinya Masuda a , Kouhei Karaike a , Tatsuro Egawa a , Taku Hamada b , Taro Toyoda c , Licht Miyamoto d , Kazuwa Nakao d , Tatsuya Hayashi a,∗ a
Kyoto University Graduate School of Human and Environmental Studies, Yoshida-Nihonmatsu-Cho, Sakyo-ku, Kyoto 606-8501, Japan Graduate School of Sport and Exercise Science, Osaka University of Health and Sport Sciences, Osaka 590-0496, Japan Division of Food Science and Biotechnology, Kyoto University Graduate School of Agriculture, Kyoto 606-8502, Japan d Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan b c
a r t i c l e
i n f o
Article history: Received 29 July 2008 Received in revised form 18 November 2008 Accepted 25 November 2008 Available online 3 December 2008 Keywords: Morus alba 5 -AMP-activated protein kinase Diabetes mellitus Glucose transport Skeletal muscle Insulin
a b s t r a c t Aim of the study: Morus alba (mulberry) leaf is a natural therapeutic agent that has been shown to have an antidiabetic effect. We explored the possibility that 5 -AMP-activated protein kinase (AMPK) is involved in metabolic enhancement by the Morus alba leaf. Materials and methods: Isolated rat epitrochlearis muscle was incubated in a buffer containing Morus alba leaf hot water extract (MLE) and the AMPK activation and related events were examined. Results: In response to MLE treatment, the Thr172 phosphorylation of the catalytic ␣ subunit of AMPK, an essential step for full kinase activation increased in a dose- and time-dependent manner. Ser79 phosphorylation of acetyl CoA carboxylase, an intracellular substrate of AMPK, increased similarly. Analysis of isoform-specific AMPK activity revealed that MLE activated both the ␣1 and ␣2 isoforms of the catalytic subunit. This increase in enzyme activity was associated with an increased rate of 3-O-methyl-d-glucose transport in the absence of insulin and with phosphorylation of AS160, a signaling intermediary leading to glucose transporter 4 translocation. The intracellular energy status, estimated from the ATP and phosphocreatine concentrations, was not affected by MLE. Conclusion: MLE stimulates skeletal muscle AMPK activity acutely without changing the intracellular energy status. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Morus alba (mulberry; family: Moraceae) is cultivated in East Asian countries, such as China, Japan, and Korea. Its leaf is mainly used in feeding silkworms (Bombyx mori L.), although it has also been used in Chinese herbal medicine for centuries. Morus alba leaf is believed to contain substances that are beneficial in preventing and alleviating “Xiao Ke” (diabetes) and is consumed as a healthpromoting drink, mulberry tea. A recent study showed that coingestion of Morus alba extract with 75 g of sucrose significantly attenuated the increase in blood glucose concentration observed over the initial 120 min of testing in nondiabetic and Type 2 diabetic individuals (Mudra et al., 2007). Morus alba leaf contains active compounds that can inhibit ␣-galactosidases, such as 1-deoxynojirimycin (Asano et al., 2001; Miyahara et al., 2004), and this effect may help
∗ Corresponding author. Tel.: +81 75 7536640; fax: +81 75 7536640. E-mail address: [email protected] (T. Hayashi). 0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.11.022
suppress postprandial hyperglycemia by reducing the rate of digestion and absorption of carbohydrates from intestines. However, intraperitoneal administration of Morus alba leaf extract has a hypoglycemic effect in streptozotocin-induced hypoinsulinemic diabetic mice (Chen et al., 1995; Kimura et al., 1995). Morus alba leaf extract also increases the rate of glucose uptake in isolated mouse diaphragm in the absence of insulin (Chen et al., 1995). These observations suggest that the acute hypoglycemic effect is caused by a direct enhancement of insulin-independent glucose transport in skeletal muscle, the major site of whole-body glucose uptake and utilization (Defronzo, 1988; Goodyear and Kahn, 1998). We hypothesized that 5 -AMP-activated protein kinase (AMPK) is involved in the antidiabetic effect of Morus alba leaf. AMPK is the major signaling intermediary in the exercise-stimulated insulinindependent glucose transport in skeletal muscle (reviewed in Kahn et al., 2005; Hardie et al., 2006; Musi and Goodyear, 2006). AMPK is a heterotrimeric kinase comprising a catalytic ␣ subunit and two regulatory subunits,  and ␥. There are two distinct ␣ isoforms in skeletal muscle: AMPK␣1 and AMPK␣2 (Stapleton et
X. Ma et al. / Journal of Ethnopharmacology 122 (2009) 54–59
al., 1996), and both isoforms are activated in response to muscle contraction, which stimulates glucose transport in the absence of insulin (Hayashi et al., 1998, 2000; Mu et al., 2001; Toyoda et al., 2006). Skeletal muscle AMPK is also implicated in a variety of antidiabetic properties of exercise, including glucose transporter 4 (GLUT4) expression (Zheng et al., 2001; Holmes et al., 2004; Nakano et al., 2006), glycogen regulation (Wojtaszewski et al., 2002; Jorgensen et al., 2004; Miyamoto et al., 2006), fatty acid oxidation (Winder and Hardie, 1996; Hutber et al., 1997; Vavvas et al., 1997), and enhanced insulin sensitivity (Fiedler et al., 2001; Buhl et al., 2002; Iglesias et al., 2002; Pold et al., 2005; Nakano et al., 2006; Tanaka et al., 2007). Skeletal muscle AMPK mediates part of glucose and lipid homeostasis by adipokines, including leptin and adiponectin, and the hypoglycemic effect of metformin (reviewed in Kahn et al., 2005; Hardie et al., 2006; Musi and Goodyear, 2006). The purpose of this study was to clarify whether Morus alba leaf acts directly on skeletal muscle to stimulate AMPK. We evaluated the effect of Morus alba leaf hot water extract (MLE) on AMPK activity in isolated rat skeletal muscle incubated in vitro. 2. Materials and methods 2.1. Experimental animals Male Wistar rats, aged 5 weeks and weighing 100–120 g, were obtained from Shimizu Breeding Laboratories (Japan). The animals were housed in an animal room maintained at 23 ◦ C with a 12:12-h light–dark cycle and fed a standard laboratory diet and water ad libitum. All protocols for animal use and euthanasia were reviewed and approved by the Kyoto University Graduate School of Human and Environmental Studies, Kyoto University Graduate School of Medicine, and Kyoto University Radioisotope Research Center in Japan. The rats were randomly assigned to the experimental groups. 2.2. Hot water extraction of Morus alba leaf Dried Morus alba leaves (product number: JAN4976015300427, manufacturing date: February 2005) were purchased from the Nakamura Chiro Association (Japan). The leaves (4 g) were powered and extracted with 50 times (w/v) of hot water (85 ◦ C) for 3 h. The extract was filtered through filter paper (No. C030633, Toyo Roshi, Japan), concentrated to a volume of 1/20 (v/v) of the initial solution volume by heating at a nonboiling temperature near to 100 ◦ C, and then dried completely under vacuum at 25 ◦ C. The dried extract (w/w = 0.59 g, yield = 15%) was used as the MLE. 2.3. Muscle incubation Rat epitrochlearis muscle was treated as described previously (Hayashi et al., 1998; Toyoda et al., 2004, 2006) with modifications. The rats were killed by cervical dislocation, and the muscles were removed rapidly. Both ends of each muscle were tied with sutures, and the muscle was mounted on an incubation apparatus with the resting tension set to 0.5 g. To recover AMPK activation during the isolating procedure, the muscle was preincubated in 7 ml of Krebs–Ringer bicarbonate buffer (KRB): 117 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2 , 1.2 mM KH2 PO4 , 1.2 mM MgSO4 , and 24.6 mM NaHCO3 containing 2 mM of pyruvate (KRBP) for 60 min. The muscle was then incubated in 7 ml of fresh buffer containing various concentrations (0–14.3 mg/ml) of MLE. The muscles were also incubated with 2 mM of 5-aminoimidazole-4-carboxamide-1-d-ribonucleoside (AICAR) for 30 min, 500 M 2,4-dinitrophenol (DNP) for 15 min or 1 M insulin for 30 min. Our previous studies have shown that these protocols elicit maximal activation of AMPK (AICAR, DNP) or glucose transport (insulin) in isolated epitrochlearis muscle (Hayashi et al., 1998, 2000). To measure the
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time course of changes in AMPK phosphorylation, the muscle was incubated with MLE (4.28 mg/ml) for up to 60 min. The buffer was bubbled with gas continuously using a mixture of 95%O2 –5%CO2 and maintained at 37 ◦ C. The muscle was then either used fresh to measure glucose uptake (see 3-O-methyl-d-glucose transport), or frozen immediately in liquid nitrogen for the subsequent measurement of isoform-specific AMPK activity and adenosine triphosphate (ATP) and phosphocreatine (PCr) concentrations, and Western blot analysis. 2.4. Western blot analysis The muscle was homogenized in ice-cold lysis buffer (1:40 w/v) containing 20 mM of Tris–HCl (pH 7.4), 1% of Triton X, 50 mM of NaCl, 250 mM of sucrose, 50 mM of NaF, 5 mM of sodium pyrophosphate, 2 mM of dithiothreitol, 4 mg/l of leupeptin, 50 mg/l of trypsin inhibitor, 0.1 mM of benzamidine, and 0.5 mM of phenylmethylsulfonyl fluoride (Buffer A), and centrifuged at 16,060 × g for 30 min at 4 ◦ C. The supernatant (10 g of protein) was mixed with loading buffer and boiled, and the denatured proteins were separated on either a 10% polyacrylamide gel for AMPK and Akt, or a 6% gel for acetyl CoA carboxylase (ACC). The proteins were then transferred to polyvinylidene difluoride membranes at 100 V for 1 h. The membrane was blocked with Block Ace (Yukijirushi Nyugyo, Japan) overnight at 4 ◦ C, and then incubated with AMPK␣ (Cell Signaling Technology, USA), phosphospecific AMPK␣ Thr172 (Cell Signaling Technology, USA), phosphospecific ACC Ser79 (Upstate Biotechnology, USA), Akt (Cell Signaling Technology, USA), and phosphospecific Akt Ser473 (Cell Signaling Technology, USA). To immunoprecipitate the Akt substrate of the 160-kDa (AS160), an aliquot of supernatant was prepared as described above (300 g of protein) and incubated with anti-AS160 (Rab-GAP) (Upstate Biotechnology, USA) and protein A-Sepharose CL4B beads (Amersham Biosciences, Sweden) overnight with end-over-end rotation at 4 ◦ C. The immunoprecipitate was washed three times in Buffer A, and then centrifuged at 4000 × g for 30 s at 4 ◦ C. The supernatant was removed, and the beads were mixed with loading buffer and boiled. The denatured proteins were then separated on a 10% polyacrylamide gel. The membrane was blocked with Block Ace overnight at 4 ◦ C, and then incubated with anti-AS160 (Rab-GAP) or antiphospho-(Ser/Thr) Akt substrate (Cell Signaling Technology, USA) as described in by Arias et al. (2007). The membrane was washed, reacted with anti-rabbit IgG coupled to peroxidase, and developed with enhanced chemiluminescence reagents according to the manufacturer’s instructions (Amersham, UK). The signals on the blot were detected and quantified using a Lumino LAS-1000 System image analyzer (Fuji Photo Film, Japan). 2.5. Isoform-specific AMPK activity assay The AMPK activity was measured as described by Hayashi et al. (2000) and Toyoda et al. (2004) with modification. Muscle was homogenized in Buffer A, as described in the Western blot analysis subsection, and the resultant supernatant liquid (100 g of protein) was immunoprecipitated with isoform-specific antibodies directed against the ␣1 or ␣2 catalytic subunit of AMPK (Toyoda et al., 2004) and protein A-Sepharose CL4B beads. The immunoprecipitate was washed twice in both Buffer A and wash buffer (240 mM of HEPES, 480 mM of NaCl). The kinase reaction was performed in 40 mM of HEPES (pH 7.0), 0.1 mM of SAMS peptide (Hayashi et al., 2000; Toyoda et al., 2004), 0.2 mM of AMP, 80 mM of NaCl, 0.8 mM of dithiothreitol, 5 mM of MgCl2, 0.2 mM of ATP (2 Ci of [␥-32 P]ATP (NEN Life Science Products, USA), in a final volume of 40 l for 20 min at 30 ◦ C. At the end of the reaction, a 15 l aliquot was removed and spotted onto Whatman P81 paper (Whatman International, UK). The filter paper was washed at least six times in 1%
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phosphoric acid and once in acetone. 32 P incorporation was quantified using a scintillation counter, and kinase activity was expressed as pmoles of incorporated ATP per mg of immunoprecipitated protein per minute. 2.6. ATP and PCr assay The frozen muscle was homogenized in 0.2 M HClO4 (3:25 w/v) in an ethanol–dry ice bath (−20 to −30 ◦ C) and centrifuged at 16,060 × g for 2 min at −9 ◦ C. The supernatant of the homogenate was neutralized with a solution containing 2 M of KOH, 0.4 M of KCl, and 0.4 M of imidazole, centrifuged at 16,060 × g for 2 min at −9 ◦ C, and then subjected to enzymatic analysis (Hayashi et al., 2000). 2.7. 3-O-methyl-d-glucose transport The assay used to measure 3-O-methyl-d-glucose (3MG) transport was performed as described by Hayashi et al. (1998) and Toyoda et al. (2004) with modification. The muscle was transferred to 2 ml of KRB containing 1 mM of 3-O-[methyl-3 H]-d-glucose (1.5 Ci/ml) (American Radiolabeled Chemicals, USA) and 7 mM d[14 C]mannitol (0.3 Ci/ml) (NEN Life Science Products, USA) at 30 ◦ C and incubated for 10 min. The muscle was blotted onto filter paper, trimmed, frozen in liquid nitrogen, and stored at −80 ◦ C. The frozen muscle was weighed and incubated in 300 l of 1 M NaOH at 80 ◦ C for 10 min. The digestate was neutralized with 300 l of 1 M HCl and the particulates were precipitated by centrifugation at 20,000 × g for 2 min. The radioactivity in aliquots of the digested protein was measured using liquid scintillation counting for dual labels, and the extracellular and intracellular spaces were calculated. 2.8. Measurement of osmolarity The osmolarity of the buffers was measured using a multisample osmometer (Osmette III, Precision Systems Inc., USA). 2.9. Statistical analysis The data are expressed as the mean ± S.E. The mean values were compared using a one-way analysis of variance (ANOVA) followed by a post hoc comparison with Fisher’s protected least-significant difference method. Two mean values ware compared using the Student’s t-test. Differences between the groups were considered significant for P < 0.05.
Fig. 1. Morus alba leaf extract (MLE) increases the phosphorylation of AMPK␣ and acetyl CoA carboxylase (ACC) in skeletal muscle. Isolated epitrochlearis muscle was preincubated for 60 min, and then incubated in the absence (basal), or presence of MLE (4.28 mg/ml) or AICAR (2 mM) for 30 min. The tissue lysate was subjected to Western blot analysis with phosphospecific antibodies to: (A) phosphorylated AMPK␣, (B) total AMPK␣, and (C) phosphorylated ACC.
2002). The marked phosphorylation of ACC paralleled the increase in AMPK phosphorylation (Fig. 1C). Similarly, AICAR stimulation caused significant phosphorylation of ACC (Fig. 1C). To determine the dose and time dependency of the AMPK phosphorylation, muscles were stimulated with MLE for various times and at various concentrations. The time course study revealed that 4.28 mg/ml of MLE induced acute phosphorylation of AMPK after an incubation period as short as 15 min, and that the peak increase occurred after an incubation period of 30–45 min (Fig. 2). The dose–response study showed a significant phosphorylation of 1.6-fold at a concentration of 4.28 mg/ml of MLE, and a 1.9-fold phosphorylation at a concentration of 14.3 mg/ml of MLE (Fig. 3). 3.2. MLE activated both AMPK ˛1 and ˛2 in skeletal muscle To identify which catalytic subunit is activated by MLE, isoform-specific AMPK activity was measured in anti-␣1 and anti-␣2 immunoprecipitates of the epitrochlearis muscle after treatment with MLE. MLE stimulation significantly increased the activity of both AMPK␣1 and AMPK␣2: the activity of AMPK␣1 = 10.5 ± 3.2 pmol/mg/min in the basal condition and
3. Results 3.1. MLE increased phosphorylation of AMPK ˛ Thr172 and ACC Ser79 in skeletal muscle The Thr172 residue in both the ␣1 and ␣2 catalytic subunits is the primary site responsible for AMPK activation (Stein et al., 2000). To determine whether MLE activates AMPK, we measured the degree of phosphorylation of AMPK ␣Thr172 using Western blot analysis employing a phosphospecific antibody. Compared with the basal condition, muscles stimulated with MLE (4.28 mg/ml, 30 min) exhibited an increased phosphorylation of Thr172 without any change in the total amount of AMPK␣ (Fig. 1A and B). The AMPK activator AICAR (2 mM, 30 min) also caused a strong phosphorylation of AMPK (Fig. 1A and B). AICAR is taken up into skeletal muscle and metabolized by adenosine kinase to form ZMP, a monophosphorylated derivative that mimics the effects of AMP on AMPK (Hardie and Carling, 1997). ACC is a downstream target of AMPK in skeletal muscle, and the phosphorylation of the Ser79 site of ACC reflects the total AMPK activity (Davies et al., 1990; Park et al.,
Fig. 2. MLE increases phosphorylation of AMPK␣ in a time-dependent manner in skeletal muscle. Isolated epitrochlearis muscle was preincubated for 60 min, and then incubated in the presence of MLE (4.28 mg/ml) for 0, 15, 30, 45, or 60 min, or AICAR (2 mM) for 30 min. The tissue lysate was subjected to Western blot analysis with phosphospecific AMPK␣ antibody. The values are expressed as the mean ± S.E., n = 4 per group. *P < 0.05 and **P < 0.01 versus the 0 min incubation group.
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Fig. 3. MLE increases phosphorylation of AMPK␣ in a dose-dependent manner in skeletal muscle. Isolated epitrochlearis muscle was preincubated for 60 min, and then incubated in the presence of 0, 1.43, 4.28, or 14.30 mg/ml of MLE for 30 min, or AICAR (2 mM) for 30 min. The tissue lysate was subjected to Western blot analysis with phosphospecific AMPK␣ antibody. The values are expressed as the mean ± S.E., n = 6 (basal, MLE) and 4 (AICAR) per group. **P < 0.01 versus the basal condition (0 mg/ml).
Fig. 5. MLE increases 3-O-methyl-d-glucose transport (3MG) transport in skeletal muscle. Isolated epitrochlearis muscle was preincubated for 60 min, and then incubated in the absence (basal), or presence of MLE (4.28 mg/ml) or insulin (1 M) for 30 min, and the 3MG transport activity was determined. The values are expressed as the mean ± S.E., n = 7 (basal, MLE) and 4 (insulin) per group. **P < 0.01 versus the basal condition.
18.7 ± 2.9 pmol/mg/min after incubation with MLE; and the activity of AMPK2␣ = 8.9 ± 1.6 pmol/mg/min in the basal condition and 24.4 ± 1.7 pmol/mg/min after incubation with MLE (Fig. 4). We used DNP, a pharmacological inhibitor of oxidative phosphorylation, as a positive control for AMPK␣1 and ␣2 activation (Hayashi et al., 2000; Fig. 4).
Akt is a key molecule in the insulin-stimulated signaling pathway leading to enhanced glucose transport and metabolism (Ueki et al., 1998). Recent studies suggest that AS160 is a downstream nexus of both Akt-mediated and AMPK-mediated glucose transport in skeletal muscle (Kramer et al., 2006; Treebak et al., 2006), and that AS160 is an intracellular substrate of AMPK in skeletal muscle (Treebak et al., 2006). In our study, in parallel with the increased 3MG transport observed in the absence of insulin (Fig. 5), MLE stimulation did not change the phosphorylation status of Akt (Fig. 6A), but significantly increased phosphorylation of AS160, as observed by AICAR stimulation (Fig. 6B).
3.3. MLE stimulated glucose transport in skeletal muscle in the absence of insulin Next, we investigated whether the activation of AMPK in skeletal muscle by MLE is associated with enhanced glucose transport. The maximally effective dose of insulin (1 M, 30 min) increased the rate of 3MG transport by 5-fold compared with the basal condition (Fig. 5). In the absence of insulin, MLE stimulation (4.28 mg/dl, 30 min) increased the rate of 3MG transport by 1.4-fold (Fig. 5).
Fig. 4. MLE activates both AMPK␣1 and ␣2 activities in skeletal muscle. Isolated epitrochlearis muscle was preincubated for 60 min, and then incubated in the absence (basal), or presence of MLE (4.28 mg/ml) for 30 min or DNP (500 M) for 15 min. The isoform-specific AMPK activity was determined in the anti-AMPK␣1 and anti-AMPK␣2 immunoprecipitates. The values are expressed as the mean ± S.E., n = 7 (basal, MLE) and 4 (DNP) per group. *P < 0.05 and **P < 0.01 versus the basal condition.
3.4. MLE treatment did not affect ATP and PCr content in skeletal muscle AMPK is activated in response to energy-consuming stress, such as muscle contraction, hypoxia, and inhibition of oxidative phosphorylation (Hayashi et al., 2000). We measured the content of ATP and PCr to determine whether MLE increases AMPK activity by energy deprivation. MLE treatment (4.28 mg/ml, 30 min) had no effect on these indicators of muscle energy status (Table 1).
Fig. 6. MLE increases phosphorylation of AS160 but not of Akt. Isolated epitrochlearis muscle was preincubated for 60 min, and then incubated in the absence (basal), or presence of MLE (4.28 mg/ml), insulin (1 M) or AICAR (2 mM) for 30 min. Tissue lysates were subjected to Western blot analysis with phosphospecific or total Akt antibodies (A). Lysates were also subjected to immunoprecipitation with AS 160 (Rab-GAP) antibody, and then subjected to Western blot analysis with antiphospho-(Ser/Thr) Akt substrate or AS 160 (Rab-GAP) antibodies (B).
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Table 1 MLE does not change the ATP and PCr content in skeletal muscle.
Basal MLE
ATP (nmol/mg)
Phosphocreatine (nmol/mg)
4.4 ± 0.2 4.0 ± 0.3
16.6 ± 0.6 16.4 ± 0.4
Isolated epitrochlearis muscle was preincubated for 60 min, and then incubated in the absence (basal), or presence of MLE (4.28 mg/ml) for 30 min, and the intracellular ATP and PCr content measured. The values are expressed as mean ± S.E.; n = 5 per group.
Table 2 Addition of MLE to KRBP does not change the osmolarity. Osmolarity (mOms/kg) Saline KRBP MLE + KRBP
284.5 ± 0.5 274.3 ± 2.9 285.3 ± 0.6
The osmolarity of saline, KRBP, and KRBP containing MLE (4.28 mg/ml) was measured with an osmometer. The values are the mean ± S.E., n = 3 per group.
3.5. MLE treatment did not change the osmolarity of KRBP Incubating rat epitrochlearis muscle in hyperosmotic buffer (KRBP with 120 mM sorbitol) activates AMPK (Hayashi et al., 2000). The osmolarity of KRBP containing MLE (4.28 mg/ml) did not differ from that of saline or KRBP (Table 2). 4. Discussion The novel finding of our study is that incubation with MLE acutely increases AMPK phosphorylation (Figs. 1–3) and activity (Fig. 4), and correspondingly, increases insulin-independent 3MG transport activity (Fig. 5) in isolated rat skeletal muscle. Enhanced phosphorylation of ACC (Fig. 1C) and AS160 (Fig. 6B), both of which are downstream targets of AMPK, indicates an actual increase in kinase activity in vivo. The phosphospecific immunoblot for Thr172 (Figs. 1A, 2, and 3), an essential site for full kinase activity, indicates that MLE induces the covalent modification by an upstream kinase. We used the isolated muscle preparation incubated in vitro to eliminate the effect of systemic confounders, such as humoral and neuronal factors and blood flow. In particular, we considered the possibility that oral administration of MLE, which may contain ␣-galactosidase inhibitors (Asano et al., 2001; Miyahara et al., 2004), could evoke secondary metabolic changes in skeletal muscle. In addition, because the isolation procedure increases AMPK␣1 activity (Toyoda et al., 2006), we exposed muscles to MLE after a preincubation period (60 min) sufficient to stabilize AMPK␣1 activity to the basal level (Toyoda et al., 2006). Thus, our method made it possible to examine the direct effect of MLE on the activity of both AMPK␣1 and AMPK␣2. We chose rat epitrochlearis muscle because it is a flat, thin muscle capable of absorbing oxygen and nutrients from a buffer in vitro (Nesher et al., 1980a,b). Differential ATPase staining has demonstrated that this muscle is a mixture of both fast- and slow-twitch fibers, i.e., more than 80% of the fibers are fast-twitch fibers and 15% are slow-twitch fibers (Nesher et al., 1980a,b). We have not yet identified the active constituent of MLE. Recent reports have suggested that berberine, a natural isoquinoline alkaloid, stimulates glucose transport by activating AMPK in L6 cultured cells (Cheng et al., 2006; Lee et al., 2006). Berberine has been isolated in variety of medical plants, such as Coptis chinensis (family: Ranunculaceae), a Chinese herb that has been used to treat diabetes for thousands of years in Asia. Berberine seems to activate AMPK by decreasing intracellular energy production because it significantly increases the ratio of AMP:ATP (Cheng et al., 2006). In support of this idea, a more recent study has suggested that berberine, and its more
biologically available derivative dihydroberberine, inhibits cellular respiration by inhibiting complex 1 of the mitochondrial respiratory chain in L6 cells (Turner et al., 2008). These observations lead us to hypothesize that berberine may be the main active constituents of MLE. However, MLE stimulation did not reduce the concentration of either ATP or PCr (Table 1), and therefore, we concluded that it is unlikely that energy-depriving compounds such as berberine play a major role in MLE-induced AMPK activation in skeletal muscle. Although we did not examine the long-term effect of MLE on skeletal muscle or whole-body metabolism, studies have clearly shown that AICAR improves the metabolic profile in animals. In our earlier study, repeated activation of AMPK by intraperitoneal injection of AICAR, three times a day for 7 d, increased GLUT4 protein by 40%, and insulin-stimulated 2-deoxyglucose transport activity by 20% in mouse soleus muscle (Nakano et al., 2006). Similarly, AICAR injections over 7 d significantly improved the response to an oral glucose tolerance test and insulin tolerance test in KKAy -CETP mice, a model of insulin-resistant type 2 diabetes mellitus (Fiedler et al., 2001). In addition, subcutaneous injections of AICAR over 7 weeks normalized the response to an oral glucose tolerance test, and decreased fasting concentrations of glucose and insulin close to the level of lean controls in obese Zucker (fa/fa) rats (Buhl et al., 2002). In ZDF rats, which are characterized by a progressive cell dysfunction and a leptin receptor defect, AICAR treatment over a period of 8 weeks increased the whole-body insulin sensitivity, which was associated with the level of muscle GLUT4 expression, and preserved the -cell function (Pold et al., 2005). Taken together, these data lead us to speculate that repeated activation of AMPK by MLE may also achieve long-term metabolic benefits that could protect against the development of diabetes. In summary, for the first time, we have demonstrated that MLE increases the activity of both AMPK␣1 and ␣2 in skeletal muscle, and that this increase is associated with insulin-independent glucose transport without change in the energy status of the muscle. We propose that Morus alba leaf fosters a metabolic milieu that reduces the risk of type 2 diabetes, at least in part, by activating skeletal muscle AMPK. Acknowledgements We are grateful to Yuichi Kawano for technical assistance, and Kaoru Ijiri and Yoko Koyama for secretarial assistance. We also thank the Radioisotope Research Center of Kyoto University for instrumental support in the radioisotope experiments. Tatsuya Hayashi was supported by research grants from the Japan Society for the Promotion of Science (17500424 and 20500576). References Arias, E.B., Kim, J., Funai, K., Cartee, G.D., 2007. Prior exercise increases phosphorylation of Akt substrate of 160 kDa (AS160) in rat skeletal muscle. American Journal of Physiology—Endocrinology and Metabolism 292, E1191–E1200. Asano, N., Yamashita, T., Yasuda, K., Ikeda, K., Kizu, H., Kameda, Y., Kato, A., Nash, R.J., Lee, H.S., Ryu, K.S., 2001. Polyhydroxylated alkaloids isolated from mulberry trees (Morusalba L.) and silkworms (Bombyx mori L.). Journal of Agricultural and Food Chemistry 49, 4208–4213. Buhl, E.S., Jessen, N., Pold, R., Ledet, T., Flyvbjerg, A., Pedersen, S.B., Pedersen, O., Schmitz, O., Lund, S., 2002. Long-term AICAR administration reduces metabolic disturbances and lowers blood pressure in rats displaying features of the insulin resistance syndrome. Diabetes 51, 2199–2206. Chen, F., Nakashima, N., Kimura, I., Kimura, M., 1995. Hypoglycemic activity and mechanisms of extracts from mulberry leaves (folium mori) and cortex mori radicis in streptozotocin-induced diabetic mice. Yakugaku Zasshi 115, 476–482. Cheng, Z., Pang, T., Gu, M., Gao, A.H., Xie, C.M., Li, J.Y., Nan, F.J., Li, J., 2006. Berberinestimulated glucose uptake in L6 myotubes involves both AMPK and p38 MAPK. Biochimica et Biophysica Acta 1760, 1682–1689. Davies, S.P., Sim, A.T., Hardie, D.G., 1990. Location and function of three sites phosphorylated on rat acetyl-CoA carboxylase by the AMP-activated protein kinase. European Journal of Biochemistry 187, 183–190. Defronzo, R.A., 1988. Lilly lecture, 1987. The triumvirate: beta-cell, muscle, liver. A collusion responsible for NIDDM. Diabetes 37, 667–687.
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