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ScienceDirect Journal of Nutritional Biochemistry 25 (2014) 136 – 143

Daidzein promotes glucose uptake through glucose transporter 4 translocation to plasma membrane in L6 myocytes and improves glucose homeostasis in Type 2 diabetic model mice Sun Hee Cheong a, b , Keisuke Furuhashi a , Katsuki Ito a , Masato Nagaoka a , Takayuki Yonezawa c , Yutaka Miura a , Kazumi Yagasaki a, c,⁎ a

Department of Applied Biological Chemistry, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan b Department of Biotechnology, Konkuk University, Chungju 380-701, Republic of Korea c Graduate School of Medicine, The University of Tokyo, Bunkyo-Ku, Tokyo 113-8654, Japan

Received 15 April 2013; received in revised form 11 September 2013; accepted 20 September 2013

Abstract Daidzein shows estrogenic, antioxidant and antiandrogenic properties as well as cell cycle regulatory activity. However, the antihyperglycemic effect of daidzein remains to be elucidated. In this study, we investigated the in vitro effect of daidzein on glucose uptake, AMPK phosphorylation and GLUT4 translocation on plasma membrane in L6 myotubes and its in vivo antihyperglycmic effect in obese–diabetic model db/db mice. Daidzein was found to promote glucose uptake, AMPK phosphorylation and GLUT4 translocation by Western blotting analyses in L6 myotubes under a condition of insulin absence. Promotion by daidzein of glucose uptake as well as GLUT4 translocation to plasma membrane by immunocytochemistry was also demonstrated in L6 myoblasts transfected with a GLUT4 cDNA-coding vector. Daidzein (0.1% in the diet) suppressed the rises in the fasting blood glucose, serum total cholesterol levels and homeostasis model assessment index of db/db mice. In addition, daidzein supplementation markedly improved the AMPK phosphorylation in gastrocnemius muscle of db/db mice. Daidzein also suppressed increases in blood glucose levels and urinary glucose excretion in KK-Ay mice, another Type 2 diabetic animal model. These in vitro and in vivo findings suggest that daidzein is preventive for Type 2 diabetes and an antidiabetic phytochemical. © 2014 Elsevier Inc. All rights reserved. Keywords: AMPK; Daidzein; Glucose transporter 4; Glucose uptake; L6 myocytes

1. Introduction Increasing incidence of diabetes mellitus is a serious world-health problem, and the International Diabetes Federation has predicted that more than 371 million people with diabetes in 2012 and that the number of diabetic patients will reach over 552 million in 2030 [1]. Especially, Type 2 diabetes mellitus is a common metabolic disease characterized by the resistance of target tissues to insulin stimulation. It is often associated with hyperglycemia, obesity, dyslipidemia, fatty liver, atherosclerosis, cancers and cardiovascular disease [2,3]. It has been well known that the isoflavones genistein and daidzein, predominantly found in soybean and soybean-derived products, are major source of phytoestrogens in human diets. Several studies in humans and animals suggest that dietary phytoestrogens play a beneficial role in reducing obesity and diabetes and improving Abbreviations: AMPK, AMP-activated protein kinase; GLUT4, glucose transporter 4. ⁎ Corresponding author. Department of Applied Biological Chemistry, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan. Fax: +81 42 367 5714. E-mail address: [email protected] (K. Yagasaki). 0955-2863/$ - see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jnutbio.2013.09.012

glucose control and insulin resistance [4]. In some animal studies, it has been reported that soy isoflavones such as genistein and daidzein maintained glycemic control, improved glucose tolerance and increased glucose transporter 4 (GLUT4) expression [5,6]. However, the molecular mechanisms underlying the metabolic action of daidzein on Type 2 diabetes mellitus have not yet been determined. In skeletal muscles, insulin resistance is a major contributor to the development of Type 2 diabetes mellitus, which is characterized by a reduced ability of insulin action to regulate blood glucose concentrations [7]. It is known that adenosine monophosphate-activated protein kinase (AMPK) exhibits a key role as a master regulator of cellular energy homeostasis. This kinase is activated in response to stresses that deplete cellular adenosin triphosphate (ATP) supplies such as low glucose, hypoxia and heat shock. Active AMPK mediates multiple beneficial effects on modulation of insulin sensitivity and energy homeostasis. Therefore, AMPK is considered to be a key therapeutic target for the treatment of Type 2 diabetes mellitus, obesity and related metabolic diseases [8]. On the other hand, glucose transporters are important regulators of metabolism, and glucose uptake is often rate limiting for cellular glucose utilization. A common feature of adipose tissue and skeletal muscle metabolism is the increase in glucose influx in response to insulin mainly mediated by

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translocation of GLUT4 from storage vesicles into the plasma membrane (PM) [9]. Among isoflavones, genistein inhibits glucose uptake in human erythrocytes via its effect on GLUT1 [10]. Genistein also inhibits GLUT4-mediated basal and insulin-stimulated glucose transport in rat adipocytes and soleus muscles [11,12]. Cederroth et al. [13] demonstrated that the dietary soy including genistein, daidzein and equol improves insulin sensitivity by increasing glucose uptake in skeletal muscle in mice. Although a number of studies concerning the therapeutic effect of daidzein have been reported, there is still little known about the potential effect of daidzein on AMPK activation and GLUT4 translocation and, hence, its effect on the blood glucose level in Type 2 diabetes mellitus. Therefore, we have examined whether or not daidzein induces glucose uptake, AMPK activation and GLUT4 translocation in vitro employing cultured muscle cells. Moreover, we investigated its effect on hyperglycemia in vivo using db/db and KK-Ay mice. 2. Materials and methods 2.1. Materials A rat skeletal muscle-derived cell line of L6 myoblasts was purchased from American Type Culture Collection (Manassas, VA; ATCC® number: CRL-1458), Dulbecco's modified Eagle medium (DMEM) was from Nissui Pharmaceutical Co. (Tokyo, Japan), fetal bovine serum (FBS) was from JRH Bioscience (Lenexa, KS, USA) and streptomycin and penicillin G were from Nacalai Tesque, Inc. (Kyoto, Japan). Bovine serum albumin (BSA) and Triton X-100 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Daidzein was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Glucose assay kit (Glucose CII Test Wako) was from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Anti-phospho-AMPKα (Thr172) and antiAMPK antibodies were obtained from Cell Signaling Technology Inc. (Beverly, MA, USA). Anti-Na+/K+-ATPase α-1 antibody was from Milipore (Billerica, MA, USA), antiGLUT4 antibody was from AbD Serotech (Oxford, UK), horseradish peroxidaseconjugated anti-mouse and anti-rabbit IgG antibodies were from Invitrogen (San Diego, CA, USA). All other chemicals were of the best grade commercially available, unless otherwise noted. Plastic multiwell plates and tubes were obtained from Nunc A/S (Roskilde, Denmark) or Iwaki brand (Asahi Glass Co., Ltd., Tokyo, Japan). 2.2. Determination of glucose uptake by cultured L6 myocytes Stock cultures of L6 myoblasts were maintained in DMEM supplemented with 10% (v/v) FBS, streptomycin (100 μg/ml) and penicillin G (100 U/ml) (10% FBS/DMEM) as described previously [14]. Effect of daidzein was examined by the procedure described previously [15] with slight modifications. Briefly, L6 myoblasts (5×104 cells/well) were subcultured into Nunc 24-place multiwell plates and grown for 11 days to form myotubes in 0.4 ml of 10% FBS/DMEM. The 11-day-old myotubes were kept for 2 h in Krebs–Henseleit buffer (pH7.4) containing 0.1% BSA, 10 mM Hepes and 2 mM sodium pyruvate (KHH buffer). The myotubes were thereafter cultured in KHH buffer containing 11 mM glucose without or with daidzein (0–100 μM) and with or without 10 μM compound C, an AMPK inhibitor, for another 4 h. Glucose concentrations in KHH buffer were determined with a glucose assay kit and a microplate reader (Appliskan, Thermo Fisher Scientific Inc., Waltham, MA, USA) at 508 nm, and the amounts of glucose consumed were calculated from the differences in glucose concentrations between before and after culture. 2.3. Preparation of PM from L6 myotubes and Western blotting L6 myoblasts (5×105 cells) were subcultured into Nunc 60 mm dishes and grown for 11 days to form myotubes in 3 ml of 10% FBS/DMEM. The 11-day-old myotubes were kept for 2 h in KHH buffer, and then, they were cultured in KHH buffer containing 11 mM glucose without or with daidzein for appropriate time intervals. PM fractions were obtained by the methods described by Nishiumi and Ashida (2007) [16] with slight modifications as described previously [17]. Cell lysate was prepared from the 11-day-old myotubes, and Western blotting for GLUT4, Na+/K+-ATPase, AMPK and phospho-AMPK was conducted as described previously [17]. 2.4. Construction of HaloTag-GLUT4 expression vector (pFN21A-rat glut4) Rat GLUT4 cDNA is amplified from rat muscle single-strand cDNA (Genostaff Co., Ltd., Tokyo, Japan) using KOD plus DNA Polymerase (Toyobo Co., Ltd., Osaka, Japan) according to the manufacturer's instructions. The primer set used for the amplification was the following: 5′-GCGCGATCGCCATGCCGTCGGGTTTCCAG-3′ and 5′GCGTTTAAACTCAGTCATTCTCATCTGGCCCTAAG-3′. The PCR product was cloned into the Sgf I/Pme I site of the expression vector pFN21A (HaloTag®7) (Promega KK, Tokyo,

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Japan). The resulting vector was designated as pFN21A-rat glut4. The construct was verified by DNA sequencing as described previously [17]. 2.5. Transfection of the expression vector into L6 myoblasts L6 myoblasts were used for the transfection and detection of pFN21A-rat glut4 because the transfection efficiency into L6 myotube was very low and we could hardly detect the HaloTag® expression (data not shown). To transfect the expression vector and control vector (pFN21A-mock), L6 myoblasts (5×104 cells/well) were cultured in a 24-well culture plate (Nunc) for glucose uptake assay or an 8-well chamber slide (Nunc) for immunocytochemistry. To support cell attachment and growth, an 8-well chamber slide was coated with collagen (Cellmatrix Type I-C, Nitta Gelatin Co., Ltd., Osaka, Japan). After 24 h, at an approximately 60% confluency (visually estimated based on viewing through a microscope), they were transfected with pFN21A-rat glut4 or pFN21A-mock using FuGENE 6 (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer's instruction. The amounts of transfected DNA were 0.3 μg/well for a 24-well culture plate and 0.2 μg/well for an 8-well chamber slide. Cells were used for the glucose uptake assay at 48 h after transfection and for immunocytochemistry at 36 h after transfection [17]. 2.6. Immunocytochemical staining At 36 h after transfection, cells were washed twice with phosphate-buffered saline (PBS) and fixed with 3.7% formaldehyde in PBS for 10 min at room temperature. After washing twice with PBS containing 0.05% Tween 20 (PBS-T), cells were blocked with 3% non-fat dried skim milk in PBS for 1 h, incubated with anti-HaloTag® rabbit polyclonal antibody (Promega KK, Tokyo, Japan) and anti-caveolin-3 goat polyclonal IgG (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) overnight at 4°C. After washing three times with PBS-T, cells were incubated with Alexa Fluor 555-conjugated anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA) and FITC-conjugated anti-goat IgG (Santa Cruz Biotechnology Inc.) for 1 h at room temperature. Finally, after washing three times with PBS-T, cells were mounted using Vectashield Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA) and examined with an Axiovert 200 M microscope (Carl Zeiss, Oberkochen, Germany). 2.7. Animals and diets All animal experiments were conducted in accordance with the guidelines established by the Animal Care and Use Committee of Tokyo University of Agriculture and Technology and were approved by this committee. Male db/db and its misty (m/ m) control (normal) mice (5 weeks of age) were obtained from Charles River Japan (Kanagawa, Japan). Male KK-Ay/Ta Jcl and C57BL/6J Jcl mice (3 weeks of age) were obtained from CLEA Japan, Inc., Tokyo, Japan. The animals were individually housed in stainless-steel cages with wire bottoms in an air-conditioned room with a temperature of 22±2°C, a relative humidity of 60±5% and an 08:00–20:00 light cycle. All the mice were maintained on a stock CE-2 pellet diet (CLEA Japan, Inc.) for 3 days and thereafter on a basal 20% casein diet (20C) for 4 days. The composition of the 20C diet was as follows (dry weight basis): 20% casein (Oriental Yeast Co., Tokyo, Japan); 7% corn oil (Hayashi Chemicals Co., Tokyo, Japan); 13.2% α-corn starch (Nihon Nosan Kogyo Co., Yokohama, Japan); 49.75% β-corn starch (Nihon Nosan Kogyo Co.); 3.5% mineral mixture (AIN-93G composition; Nihon Nosan Kogyo Co.); 1% vitamin mixture (AIN-93 composition; Nihon Nosan Kogyo Co.); 0.25% choline bitartrate (Wako Pure Chemical Industries, Ltd.); 0.3% L-cystine (Wako Pure Chemical Industries, Ltd.) and 5% cellulose powder (Oriental Yeast Co.). After preliminary feeding for 1 week, blood was collected from the tail vein at 12:00 under the conditions that the mice were deprived of their diet at 9:00 (db/db mice) or allowed free access to their diet and water without diet deprivation (KK-Ay mice). After bursting of the blood cells (5 μl) in water (45 μl), 20% (wt/vol) trichloroacetic acid aqueous solution (50 μl) was added, and the test tube containing the mixture was kept in ice-cold water. The mixture was then centrifuged at 12,000 rpm (MX-160, Tomy Seiko Co., Ltd., Tokyo, Japan) and 4°C for 5 min. The resultant supernatant (10 μl) was subjected to glucose determination using the Glucose CII Test Kit and measurement of the absorbance at 508 nm with a microplate reader (Appliskan, Thermo Fisher Scientific Inc.). Subsequently, the db/db mice (6 weeks of age) or KK-Ay mice (4 weeks of age) were divided into two groups with similar fasting or nonfasting blood glucose levels and body weights (0 week). The db/ db or KK-Ay mice in each of the two groups were given either the 20C diet as a diabetic control group or the 20C diet supplemented with 0.1% daidzein as a daidzein-fed diabetic group for 4 or 5 weeks. Daidzein was supplemented to the 20C diet at the expense of the β-cornstarch. Likewise, the normal mice were given the 20C diet as a nondiabetic group for 4 or 5 weeks. Water and each diet were always available, and blood was collected every week at 12:00 h to determine the fasting (db/db mice) or non-fasting (KK-Ay mice) blood glucose levels as described above. Glucose excretion into urine of the KK-Ay/Ta Jcl mice was also determined every week using a commercial urine test paper (URIACE-M, Terumo Corporation, Tokyo, Japan). At the end of feeding period, blood was collected at 12:00 from the tail vein, followed by exsanguination from the heart under anesthesia with Somnopentyl (Kyoritsu Seiyaku Corp., Tokyo, Japan). Liver and gastrocnemius muscle were excised immediately, rinsed, blotted on filter paper, weighed, frozen in liquid nitrogen and stored at −85°C until analyses.

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2.8. Biochemical analyses of blood and liver

2.10. Statistical analyses

Blood glucose, serum and hepatic triglyceride (TG), serum total cholesterol (T-Ch) and lipid peroxide (thiobarbituric acid-reactive substances, TBARS) were measured with commercial kits (Wako Pure Chemical Industries and other suppliers in Japan). Serum insulin, adiponectin and tumor necrosis factor-α (TNF-α) were measured by immunoassays using commercial ELISA kits. The homeostasis model assessment (HOMA) index was calculated from the fasting blood glucose and insulin levels as a measure of insulin resistance as follows: HOMA=fasting glucose level (mg/dl)×fasting insulin level (ng/ml)/405.

All data are expressed as means±standard errors of the means (S.E.M.). Multigroup comparisons were carried out by one-way analysis of variance followed by Tukey–Kramer multiple comparisons test. Values of Pb.05 were considered statistically significant.

3. Results

2.9. Tissue extracts and Western blotting

3.1. Effect of daidzein on glucose uptake, AMPK phosphorylation and GLUT4 translocation in L6 myotubes

Protein isolation and Western blotting were performed, as described previously [18], with slight modifications. Briefly, the skeletal muscle was homogenized in a Polytron homogenizer (Polytron CH-6010, Kinematica GmbH, Luzern, Switzerland) using the homogenization buffer (50-mM Tris, 0.1% sodium dodecyl sulfate (SDS), 150-mM sodium chloride, 1% Triton X-100, 1% sodium deoxycholate, 1-mM EDTA and 1-mM sodium orthovanadate) at 4°C, and the supernatant obtained was again centrifuged at 12,000 rpm (MX-160, Tomy Seiko Co., Ltd.) for 20 min at 4°C. The resulting supernatant was retained as the total cell lysate, and the pellet obtained was resuspended in 0.5 ml of buffer and used as the membrane fraction. Muscle homogenate containing 100 μg protein was conducted the Western blotting for AMPK and phospho-AMPK as described previously [17].

To investigate mechanisms for daidzein actions, we first examined its effect on glucose uptake by L6 myotubes in vitro. Daidzein dosedependently and significantly increased glucose uptake at concentrations of 25–100 μM in the absence of insulin (Fig. 1A left). Also, we performed glucose uptake assay using compound C, an inhibitor of AMPK, to determine the regulatory mechanism by which daidzein induced the glucose uptake in L6 myotubes. The promotion of glucose uptake by daidzein was significantly inhibited by treatment with compound C (Fig. 1A right). These results suggest that the stimulatory

Fig. 1. Effect of daidzein on glucose uptake, AMPK phosphorylation and GLUT4 translocation in L6 myotubes. (A) Glucose uptake for 4 h was measured in 11-day-old L6 myotubes. The myotubes were kept for 2 h in Krebs–Henseleit buffer (pH 7.4) containing 0.1% BSA, 10 mM Hepes and 2 mM sodium pyruvate (KHH buffer), and then they were cultured in KHH buffer containing 11 mM glucose without or with daidzein (0, 25, 50 and 100 μM) and 10 μM compound C. Each value represents the mean±S.E.M. for six wells. Values not sharing a common letter are significantly different at Pb.05 by Tukey–Kramer multiple comparisons test. (B) Effect of daidzein (100 μM) on AMPK phosphorylation in cultured L6 myotubes. L6 myotubes were incubated in KHH buffer without glucose for 2 h. They were then incubated in KHH buffer containing 11 mM glucose in the presence or absence of 100 μM daidzein and 1 mM AICAR for indicated time intervals. Total lysates were analyzed by Western blotting with anti-AMPK and anti-phospho-AMPK antibodies. Protein bands were quantified by ImageJ, and ratios of p-AMPK/AMPK are shown. The ratio at 0 min is regarded as 1. (C) Effect of daidzein on GLUT 4 translocation in cultured L6 myotubes. L6 myotubes were incubated for 15 min in KHH buffer containing 100 μM daidzein with 11 mM glucose. Membrane fractions of L6 myotubes were prepared by a rapid PM preparation method. The PM fractions (20 μg) were subjected to SDS-PAGE and Western blotting analyses using anti-GLUT4 and anti-Na+/K+-ATPase antibodies. Protein bands were quantified by ImageJ and ratios of GLUT4/ATPase in PM fraction are shown.

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effect of daidzein on glucose uptake is dependent on the AMPK pathway. Phosphorylation ratio of AMPK commenced to increase 5 min after daidzein (100 μM) treatment. Maximum phosphorylation ratio of AMPK was attained 60 min after treatment, and thereafter, phosphorylation ratio of AMPK gradually diminished up to 240 min (Fig. 1B). It is known that AMPK phosphorylation causes GLUT4 translocation to PM. Thus, we examined whether or not daidzein would promote GLUT4 translocation to PM by Western blotting analyses using antibodies to GLUT4 and Na+/K+-ATPase. Treatment of L6 myotubes with 100 μΜ daidzein for 30 min promoted GLUT4 translocation. Moreover, the ratio of GLUT4 to Na+/K+-ATPase, a PM marker enzyme, was found to increase in PM fractions of daidzeintreated L6 myotubes (Fig. 1C).

3.2. Bioimaging of GLUT4 translocation in L6 myoblasts transfected with pFN21A-rat glut4 vector We examined the effect of daidzein on glucose uptake and cellular GLUT4 localization by bioimaging in cultured L6 myoblasts transfected with pFN21A-mock vector or pFN21A-glut4 vector. In this study, 100 μΜ of daidzein treatment markedly increased the glucose uptake in HaloTag®-GLUT4 expressed L6 myoblasts (Fig. 2A). Fig. 2B (left) shows cellular localization of HaloTag protein alone (Mock DAI(−) and Mock DAI(+)) and HaloTag-GLUT4 protein (GLUT4 DAI(−) and GLUT4 DAI(+)). Fig. 2B (center) shows cellular localization of caveolin-3, a biomembrane marker of muscle cells, and Fig. 2B (right) shows their merging. In cells transfected with pFN21A-mock vector, HaloTag protein and caveolin-3 were expressed in the whole area except for nuclear compartment but did not colocalize (Mock DAI(−) and Mock DAI(+)). Likewise, HaloTag protein and caveolin-3 were expressed similarly but more strongly expressed in cells transfected with pFN21A-GLUT4 vector than in cells transfected with pFN21A-mock vector, and co-localization of two proteins was recognized. Daidzein treatment for 30 min strengthened their co-localization in the PM compartment as shown by yellowish

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color (GLUT4 DAI(−) vs. GLUT4 DAI(+)). These findings based on bioimaging method support the promotive effect of daidzein on GLUT4 translocation to PM that was demonstrated in biochemical analysis (Fig. 1C).

3.3. Effect of daidzein on biochemical parameters in db/db mice To investigate the in vivo effect of daidzein, we employed db/db mice, a severe Type 2 diabetic animal model. As shown in Table 1, serum glucose level and HOMA index of the control group were markedly increased as compared with those of the normal group. In contrast, daidzein significantly suppressed the rises in the serum glucose level and HOMA index compared to the control group. Serum T-Ch concentration of the control group was significantly increased as compared with that of the normal group. Daidzein, however, significantly suppressed the rise in the serum T-Ch concentration. Serum TNF-α and TBARS concentrations in the control group were significantly increased as compared with those in the normal group. These concentrations in the daidzein group tended to decrease as compared with those in the control group.

3.4. Effect of daidzein on blood glucose level and AMPK phosphorylation in skeletal muscle of db/db mice Fig. 3 shows the effect of daidzein on fasting blood glucose level and AMPK phosphorylation in skeletal muscle in db/db mice. In this study, daidzein (0.1% in the diet) significantly suppressed the rises in the fasting blood glucose level after 1, 2 and 3 weeks of feeding compared to the control group (Fig. 3A). Fig. 3B shows the effect of daidzein on AMPK phosphorylation in skeletal muscle of db/db mice. Daidzein treatment led to significant increase in AMPK phosphorylation compared to the control group. In this study, the food intake of the daidzein group for 3 weeks did not differ significantly from that of the control group, suggesting that the suppressive effect of daidzein

Fig. 2. Effect of daidzein on glucose uptake and cellular GLUT4 localization by bioimaging in cultured L6 myoblasts transfected with pFN21A-mock vector or pFN21A-glut4 vector. (A) Effect of daidzein on glucose uptake in L6 myoblasts expressing proteins of HaloTag or HaloTag-GLUT4. L6 myoblasts were exposed to 0 μM (−) or 100 μM (+) daidzein (DAI(−) or Dai(+)) for 4 h. Each value represents the mean±S.E.M. for four wells. Values not sharing a common letter are significantly different at Pb.05 by Tukey–Kramer multiple comparisons test. (B) L6 myoblasts stably expressing proteins of HaloTag or HaloTag-GLUT4 were processed for immunocytochemistry using anti-HaloTag antibody and anti-caveolin-3 antibody. Cellular localization of HaloTag and HaloTag-GLUT4 is shown in red fluorescence (left) and that of caveolin-3 is shown in green fluorescence (center). Merge image is also shown in yellow fluorescence (right). L6 myoblasts were exposed to daidzein (0 or 100 μM) for 30 min.

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Table 1 Metabolic characteristics of mice fed a standard diet or daidzein-containing diet for 4 weeks in db/db mice Measurement

NOR

CNT a

Body weight gain (g/28 days) 3.7±0.1 Initial fasting blood glucose (mg/dl) 167.9±3.8 Serum TNF-α (pg/ml) 22.0±4.3a Serum adiponectin (μg/ml) 13.6±0.6a Serum glucose (mg/dl) 121.3±15.9a Serum insulin (ng/ml) 1.2±0.3a HOMA index 0.4±0.1a Serum lipid levels Total cholesterol (mg/dl) 182.7±11.6a Triglyceride (mg/dl) 79.5±12.3 TBARS (nmol/ml) 3.8±0.8a Hepatic lipid levels Total cholesterol (mg/whole liver/100 g 5.4±0.7a B.W) Triglyceride (mg/whole liver/100 g B.W) 53.9±5.3a

DAIDZEIN

11.4±0.7 13.2±1.1b 131.0±17.5 134.5±10.7 136.8±29.5b 67.6±15.3ab 7.8±0.3b 7.2±0.3b 701.8±52.3b 526.4±56.2c 6.9±1.0b 5.8±1.6b 12.0±2.0b 6.8±1.5c

concentration. The control group showed a more acute increase in glucose excretion in proportion to the non-fasting glucose level than did the daidzein-treated group (Fig. 4B).

b

366.1±6.3b 96.6±12.4 8.2±1.0b

320.1±11.1c 73.4±10.7 7.4±1.3ab

14.0±1.8b

14.5±1.0b

309.0±40.8b 343.2±32.5b

Each value represents the mean±S.E.M. for six db/db mice. Values not sharing a common letter are significantly different at Pb.05 by Tukey–Kramer multiple comparisons test.

on the fasting blood glucose level was not caused by reduced food intake but instead by its pharmacological action.

3.5. Effect of daidzein on non-fasting blood glucose level and urinary glucose excretion in KK-Ay/Ta Jcl mice Fig. 4 shows the effect of daidzein on the non-fasting blood glucose level and urinary glucose excretion in KK-Ay/Ta Jcl mice. The nonfasting blood glucose level in KK-Ay/Ta Jcl mice gradually increased for 3 weeks and thereafter maintained high values up to 5 weeks in the control group. On the other hand, daidzein (0.1% in the diet) significantly suppressed the rises in the non-fasting blood glucose level after 1 and 3 weeks of feeding compared to the control group (Fig. 4A). To investigate renal glucose excretion as a function of the blood glucose concentration in KK-Ay/Ta Jcl mice, we measured the urinary glucose concentration simultaneously with the blood glucose

4. Discussion Insulin resistance in skeletal muscle is related to the development of metabolic diseases such as hyperglycemia and obesity. Some previous studies reported that treatment of isolated rodent muscle with AICAR, a pharmacological agent that activates AMPK, is associated with an increase in glucose transport by an insulinindependent pathway, and this is related to increased translocation of the insulin-sensitive glucose transporter GLUT4 from intracellular microvesicles to membrane of skeletal muscle [19,20]. Recently, several studies have attempted to discover natural bioactive compounds for the improvement of insulin resistance associated with Type 2 diabetes mellitus [21]. Especially, some researchers have focused on isoflavones such as genistein, daidzein and glycitein as important bioactive components of soybean [22]. Several animal and human studies have also provided evidence that consumption of these isoflavones alleviates some of the symptoms associated with Type 2 diabetes such as insulin resistance and glycemic control [23–25]. A previous in vitro study reported that isoflavones possess antidiabetic effect by α-glucosidase inhibitory activity [26]. On the other hand, it has been reported that the metabolic features of ob/ob mice are similar to the pathogenesis of Type 2 diabetes in humans [27]. In this study, therefore, we investigated the antihyperglycemic activity of daidzein in db/db mice, a similar Type 2 diabetic animal model, and clarified the regulatory mechanism of glucose uptake by daidzein from a viewpoint of GLUT4 translocation and AMPK activation in cultured L6 myotubes. In this study, we investigated the signaling pathway for glucose uptake by daidzein using AMPK inhibitor in L6 myotubes and found that the stimulatory effect of daidzein on glucose uptake appeared to be dependent on the AMPK pathway. In our previous works, we confirmed that genistein regulated glucose uptake by increasing the phosphorylation of AMPK and induction of GLUT4 translocation in L6

Fig. 3. Effect of daidzein on (A) fasting blood glucose levels and (B) AMPK phosphorylation in gastrocnemius muscle of db/db mice. (A) The db/db mice were fasted for 3 h before the collection of blood. Blood samples were collected from tail vein. Each value represents the mean±S.E.M. of six mice. Values not sharing a common letter are significantly different at Pb.05 by Tukey–Kramer multiple comparisons test. (B) Homogenates of gastrocnemius muscle were subjected to SDS-PAGE and Western blotting analysis using anti-phospho-AMPK and anti-AMPK antibodies. Each value represents the mean±S.E.M. of three mice. Values not sharing a common letter are significantly different at Pb.05 by Tukey–Kramer multiple comparisons test.

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Fig. 4. Effect of daidzein on nonfasting blood glucose levels and urinary glucose excretion in KK-Ay/Ta Jcl mice. (A) KK-Ay/Ta Jcl mice were kept on diets during blood collection from tail vein. Each value represents the mean±S.E.M. of six mice. Values not sharing a common letter are significantly different at Pb.05 by Tukey–Kramer multiple comparisons test. (B) Urinary glucose excretion in mice was determined using urine test paper. Each group consisted of six mice.

myotubes [28]. The present results indicated that daidzein, like genistein, stimulated glucose uptake in L6 myotubes by increasing GLUT4 translocation to the PM via AMPK activation. Thus, the stimulatory mechanism of daidzein and genistein for glucose uptake in L6 myotubes is considered to be similar. In our separate experiments, 17β-estradiol was demonstrated to exert no influence on glucose uptake in L6 myotubes, and ICI182780, an estrogen receptor antagonist, failed to cancel the promoting effect of glucose uptake by genistein in L6 myotubes (unpublished observation). These findings indicate under our experimental conditions that glucose uptake in L6 myotubes is not regulated by estrogen. They also indicate that genistein and daidzein do not act as phytoestrogens, at least, in the myotubes. More recently, it has been reported that natural compounds promote glucose uptake in L6 myotubes, activate AMPK and suppress the rises in blood glucose concentrations in Type 2 diabetic model mice [15,17,36]. In our in vivo study, we found that daidzein markedly suppressed the rises in the serum glucose level and HOMA index, a useful clinical index of insulin resistance, as well as serum T-Ch level. Others have reported that isoflavone supplementation can decrease serum T-Ch and serum low-density lipoprotein (LDL)-Ch levels in mice and humans [29,30]. Jayagopal et al. reported that HOMA index was significantly reduced in postmenopausal women with dietcontrolled Type 2 diabetes who consumed soy protein isolate for 12 weeks [23]. Our data further confirmed that daidzein significantly

suppressed the rises in the fasting blood glucose level compared with the diabetic group after 1, 2 and 3 weeks in db/db mice. Similar to our results, it was reported that genistein and daidzein supplementation lowered the blood glucose levels significantly without changes of plasma insulin and C-peptide levels [6]. In the present study, we also confirmed that daidzein treatment led to a markedly increase in AMPK phosphorylation of gastrocnemius muscle compared to the control group in db/db mice as well as in cultured L6 myotubes. Recently, Kim et al. have also reported that genistein can activate AMPK in mice fed with a high-fat diet [31]. In general, KK-Ay/Ta Jcl mice have been used as Type 2 diabetic model animals that rapidly develop peripheral glomerular capillary basement membrane thickening in the kidney, resulting in severe hyperinsulinemia and hyperglycemia [32]. The KK-Ay/Ta Jcl mice were found to rapidly develop hyperglycemia in this study. Nevertheless, daidzein significantly suppressed the increase in the nonfasting blood glucose level after 1 and 3 weeks of feeding compared with the control group. We also found a delay in renal glucose secretion in the daidzein-fed KKAy/Ta Jcl mice compared with the control group. In our previous study, we have already confirmed that genistein decreased nonfasting blood glucose levels in KK-Ay/Ta Jcl mice [28]. Taken together, these findings support the antihyperglycemic effect of daidzein in vivo as well as in vitro. Generally, AMPK has been reported as a suppressor of hepatic gluconeogenesis [33]. Interestingly, we have already verified in this

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study that daidzein phosphorylated AMPK in cultured L6 myotubes and in gastrocnemius muscle of db/db mice. Hyperglycemia in Type 2 diabetes is characterized by enhanced glucose production in the liver [34,36]. To investigate hepatic gluconeogenesis, we examined expression of PEPCK and G6Pase in the liver. PEPCK and G6Pase are two rate-limiting enzymes in the gluconeogenic process in hepatocytes. In this study, hepatic mRNA expression levels of PEPCK and G6Pase tended to decrease by daidzein treatment (data not shown). Similarly, Park et al. [6] reported that soy isoflavone including genistein and daidzein modulate hepatic glucose and lipid regulatory enzyme activities in db/db mice. It was also reported that genistein and daidzein regulated the glucose homeostasis in Type 1 diabetic mice by down-regulating enzyme activities such as G6Pase, PEPCK and CPT [35]. This may be a new possible mechanism for daidzein action in the regulation of glucose metabolism. Resveratrol was found to activate AMPK in muscle cells [37]. However, the precise mechanism by which resveratrol activates AMPK remains to be elucidated. In particular, the direct molecular target of resveratrol has been elusive [38]. Park et al. recently show that resveratrol directly inhibits cAMP-dependent phosphodiesterases (PDEs) [39]. They have reported that the metabolic effects of resveratrol result from competitive inhibition of cAMP-degrading PDE, leading to elevated cAMP levels. The resulting activation of Epac1, a cAMP effector protein, increases intracellular Ca2+ levels and activates the CamKK-AMPK pathway via phospholipase C and the ryanodine receptor Ca2+-release channel. As a consequence, resveratrol increases AMPK phosphorylation, NAD+ and the activity of Sirt1. Inhibiting PDE4 with rolipram reproduces all of the metabolic benefits of resveratrol, including prevention of diet-induced obesity and an increase in mitochondrial function, physical stamina, and glucose tolerance in mice. Therefore, administration of PDE4 inhibitors may also protect against and ameliorate the symptoms of metabolic diseases associated with aging [39]. It is likely that other phytochemicals also activate AMPK by inhibiting PDEs because many of them have been shown to be PDE inhibitors [40]. Thus, it seems worthy from the aspect of direct target identification to examine whether or not daidzein as well as genistein could inhibit PDEs. In our present study, the average amount of daidzein ingested from diet containing 0.1% daidzein was calculated to be ca. 100 mg/kg body weight/day/mouse. Daidzein (100 mg/kg) has a potential to adversely affect erectile function in a dose- and time-related manner in rats, if consumed for a long period [41]. In reproductive safety studies with genistein in rats, the no observed adverse effect level (NOAEL) for maternal toxicity and adverse effects on embryonic development was considered to be 100 mg/kg when administered orally by dietary administration [42]. In subchronic and chronic safety studies with genistein in dogs, the NOAEL was considered to be 100 mg/kg [43]. Park et al. reported that the blood glucose and HbA1c levels in db/db mice were significantly lower in the 0.02% genisteinand 0.02% daidzein-containing diets-fed groups than in the controldiet group [6]. Further studies on antidiabetic effect of daidzein at lower doses of, for instance, 0.05% or 0.02% in diet are required in Type 2 diabetic animal models, ob/ob and KK-Ay mice as well as db/db mice. According to Reagan-Shaw et al., effective doses in humans are suggested to be one eighth to one twelfth of those in mice [44]. Although the necessary amounts of daidzein to apply its hypoglycemic effect in human body have not been established yet, our research from the new aspect of action mechanism for daidzein provides the importance and basis of its glucose-lowering effect for further researches in humans. In summary, we have found for the first time that one of the modes of daidzein action is activation of AMPK followed by GLUT4 translocation to PM of muscle cells that are main sites where glucose uptake occurs. We also confirmed that daidzein significantly suppressed the rises in blood glucose levels in db/db and KK-Ay

mice. These results suggested that daidzein has an antihyperglycemic effect as a novel therapeutic candidate, and this may be mediated by a main mechanism including AMPK activation and induction of GLUT4 translocation.

Acknowledgments This work was supported in part by a grant from the Japan Society for the Promotion of Science and in part by a grant from the Takano Life Science Research Foundation, Ibaragi, Japan. Authors declare no conflict of interest.

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