Journal of Functional Foods 38 (2017) 438–446

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Journal of Functional Foods

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An isoflavone compound daidzein elicits myoblast differentiation andmyotube growth

https://doi.org/10.1016/j.jff.2017.09.0161756-4646/� 2017 Elsevier Ltd. All rights reserved.

Abbreviations: BS Ext, Black soybean extract; DEX, dexamethasone; DM,differentiation medium; DMEM, Dulbecco modified Eagle’s medium; ER, estrogenreceptors; ERE, estrogen response element; FBS, fetal bovine serum; GLUT4, glucosetransporter 4; GM, growth medium; mTOR, mammalian target of rapamycin; MHC,myosin heavy chain; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel elec-trophoresis; SRC-1, steroid hormone receptor coactivator; Stx4, Syntaxin 4.⇑ Corresponding author.

E-mail address: gbae@sookmyung.ac.kr (G.-U. Bae).1 These authors are equally contributed in this study.

Sang-Jin Lee a,1, Tuan Anh Vuong b,1, Ga-Yeon Go a, Yoo-Jin Song a, Seulah Lee c, Soo-Yeon Lee a,Shin-Woong Kim a, Jaecheol Lee d, Yong Kee Kim a, Dong-Wan Seo e, Ki Hyun Kim c, Jong-Sun Kang b,Gyu-Un Bae a,⇑aResearch Center for Cell Fate Control, College of Pharmacy, Sookmyung Women’s University, Seoul 140-742, Republic of KoreabDepartment of Molecular Cell Biology, Single Cell Network Research Center, Sungkyunkwan University School of Medicine, Suwon 440-746, Republic of Koreac School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of KoreadDivision of Cardiology, Department of Medicine, Stanford University School of Medicine, CA 94305-5454, USAeCollege of Pharmacy, Dankook University, Cheonan 330-714, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history:Received 25 February 2017Received in revised form 24 August 2017Accepted 12 September 2017

Keywords:Akt signalingDaidzeinMuscle regenerationMuscular atrophyMyoblast differentiationMyoD

Preservation of muscle stem cell function is critical for muscle homeostasis and the regenerative capacityof stem cells declines with age, leading to muscle loss. Previously, daidzein, a natural isoflavone fromLeguminosae, has been shown to prevent TNF-a-induced muscle atrophy. However, the molecular mech-anisms of daidzein in these processes are currently unknown. In this study, we demonstrate a positiveeffect of black soybean extract (BS Ext) and daidzein on myoblast differentiation and myotube growth.Daidzein prevents dexamethasone-induced myotube atrophy through Akt activation. The promyogeniceffect of BS Ext and daidzein is mediated through two promyogenic kinases, Akt and p38MAPK that inturn activates a key myogenic transcription factor MyoD. Additionally, BS Ext and daidzein promote myo-tube growth through Akt/mTOR/S6K pathway. Finally, daidzein augments MyoD-dependent myogenicconversion of mouse embryonic fibroblasts. Taken together, our data suggest that daidzein promotesmyogenic differentiation and myotube hypertrophy through p38 and Akt/mTOR/S6K pathway.

� 2017 Elsevier Ltd. All rights reserved.

1. Introduction

Skeletal muscle weakness and loss occurring in aging or otherpathological conditions, such as diabetes, chronic inflammationor cancers has been shown to exacerbate metabolic syndromes,ultimately leading to declined life quality and lifespan (Sanchezet al., 2013). Skeletal muscle maintenance is controlled by the bal-ance action between protein synthesis and degradation in musclesand the condition associated decreased protein synthesis andincreased degradation causes muscle atrophy (Goldspink,

Morton, Loughna, & Goldspink, 1986). In contrast, skeletal musclehypertrophy is associated with increased size of pre-existing skele-tal muscle fibers accompanied by incremented protein synthesis(Sanchez et al., 2013). In addition, unperturbed muscle stem cellfunction is also critical for maintenance of muscle mass and func-tion and the impaired stem cell function has been also implicatedin muscle weakness and loss (Motohashi & Asakura, 2014). The factthat skeletal muscle is the largest energy consuming organ, muchattention has been paid to understand molecular regulatory path-ways controlling muscle stem cell function and muscle growth aswell as to identify compound(s) to improve muscle function andmass.

Daidzein is a natural isoflavone from Leguminosae and struc-turally similar to natural and synthetic estrogen and antiestrogen(Kuiper et al., 1997; Sun et al., 2016). Daidzein can bind to estrogenreceptors (ER) (Kuiper et al., 1997) and has been shown to activatethe classic estrogen response element (ERE) pathway throughincreasing the expression of ERa, ERb and steroid hormone recep-tor coactivator (SRC-1) (Sun et al., 2016). The beneficial effects of

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daidzein have been reported in various systems, including osteo-blast cells, neuronal cells, cardiomyocytes and skeletal muscle cells(Ahmed et al., 2017). In H9C2 cardiomyocytes, daidzein protectscells from the isoproterenol-induced apoptosis through Akt activa-tion (Hu et al., 2016). Furthermore, daidzein improves insulin sen-sitivity in skeletal muscle by increasing glucose transporter 4(GLUT4) expression and glucose uptake (Cederroth et al., 2008).Recently, daidzein significantly suppresses MuRF1 promoter activ-ity and prevents TNF-a-induced myotube atrophy through SIRT1activation (Hirasaka et al., 2013). However, the effects of daidzeinon myoblast differentiation and myotube growth have not beenexamined.

In this study, we demonstrate that BS Ext and daidzeinenhances myoblast differentiation and myotube growth via activa-tion of promyogenic signaling pathways. Thus, daidzein has apotential for a therapeutic or neutraceutical remedy to intervenemuscle weakness and atrophy.

2. Material and methods

2.1. Reagents

Daidzein (PubChem CID: 5281708) was purchased from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum (FBS) and Dulbeccomodified Eagle’s medium (DMEM) were purchased from ThermoScientific (Waltham, MA). Horse serum (HS) are obtained fromWelGene (Daegu, Korea). Antibodies used in this study were as fol-lowing: MHC (MF-20; the Developmental Studies Hybridoma Bank,Iowa, IA), phospho-p38MAPK (recognizing phospho-T180/-Y182sites), Akt, phospho-Akt (recognizing the phospho-S413 site),mTOR, phospho-mTOR, p70S6K, phospho-p70S6K (Cell SignalingTechnology, Beverly, MA), p38MAPK, MyoD, Myogenin, E2A, a-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA) and pan-Cadherin (Sigma-Aldrich). Lipofectamin 2000 was obtained fromInvitrogen (Carlsbad, CA). SB203580 and LY294002 were obtainedfrom CalBiochem (La Jolla, CA). Complete proteinase inhibitorcocktail and protein G agarose beads were from Roche Diagnostics(Basel, Switzerland). DEX and all other chemicals were obtainedfrom Sigma-Aldrich.

2.2. Preparation of the ethanolic extract of black soybean (BS Ext) andLC/MS analysis

Black soybean was extracted using 80% ethanol (v/v) at roomtemperature. To remove the solvents in the extract it was sub-jected to a rotary evaporator at 42 �C under reduced pressureand followed by freeze-dry using a lyophilizer. The ethanolicextract of black soybean (BS Ext) was analyzed by LC/MS (AgilentTechnologies, Santa Clara, CA). Stock solution of the ethanolicextract was prepared by dissolving 1 mg of sample in 200 lLmethanol. The solution was further diluted with methanol to pro-vide a solution of 50 lg/mL. It was then filtered through a 0.45 mmhydrophobic PTFE filter and finally analyzed by LC/MS Agilent 1200Series analytical system equipped with a photodiode array (PDA)detector combined with a 6130 Series ESI mass spectrometer.Analysis was performed by the injection of 7 lL of the sampleusing a Kinetex C18 column (2.1 � 100 mm, 5 lm; Phenomenex,Torrance, CA) set at 25 �C. The mobile phase consisting of formicacid in H2O [0.1% (v/v)] (A) and methanol (B) was delivered at aflow rate of 0.3 mL/min by applying the following programmedgradient elution: 0–50% (B) for 30 min, 50–100% (B) for 1 min,100% (B) isocratic for 10 min, and then 0% (B) isocratic for10 min, to perform post-run reconditioning of the column. TheLC/MS data in positive and negative ion modes and comparisonof in-house UV database and literature data allowed the identifica-

tion of compounds 1–7 [daidzin (1), glycitin (2), genistin (3),malonyldaidzin (4), malnylglycitin (5), malonylgenistin (6), anddaidzein (7)] (Kim et al., 2016), which indicates the profiling ofthe BS Ext.

2.3. Cell cultures

Myoblast C2C12 cells, embryonic fibroblast 10T1/2 cells andembryonic kidney 293 T cells were cultured as described previ-ously (Bae et al., 2009, 2010). To induce differentiation of C2C12myoblasts, cells at near confluence were switched from DMEMcontaining 15% FBS (growth medium, GM) to DMEM containing2% HS (differentiation medium, DM) and myotube formation wasobserved normally at approximately 2–3 days of differentiation.The efficiency of the myotube formation was quantified by a tran-sient differentiation assay as previously described (Lee et al., 2016).For the DEX-induced atrophy study, C2C12 cells were induced todifferentiate in DM for 1 day and treated with 400 ng/mL DEXand 50 nM daidzein followed by incubation in DM for 2 days(Stitt et al., 2004). For our experiments that involved p38MAPKinhibitors and Akt inhibitors, C2C12 cells were treated with50 nM daidzein after pre-incubation with 2.5 lM SB203580 or1 lM LY294003 in fresh culture medium for 30 min, respectively.For hypertrophic myoblast differentiation, C2C12 cells wereinduced to differentiation for 2 days and then treated with 50 nMdaidzein for additional 2 days in DM. For the trans-differentiationstudy, 10T1/2 cells were cultured in DMEM supplemented with10% FBS in 100 mm plates, and transfected with 5 lg of MyoD orcontrol vector (pBp). After 1–2 day incubation, 10T1/2 cells weretreated with 50 nM daidzein in 2% HS for 2 days.

2.4. Western blotting analysis and immunoprecipitation analysis

Western blot analysis was performed as previously described(Bae et al., 2010). Briefly, cells were lysed in cell lysis buffer(10 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) containing complete proteinase inhibitor cocktail and sodiumdodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)was performed. The primary antibodies used were anti-pan-Cadherin, anti-p38MAPK, anti-phospho-p38MAPK (the phosphory-lated, active form of p38MAPK), anti-Akt, anti-phospho-Akt (thephosphorylated, active form of Akt), anti-mTOR, anti-phospho-mTOR, anti-p70S6K, anti-phospho-p70S6K, anti-MHC, anti-Myogenin, anti-MyoD, anti-E2A and anti-a-tubulin.

For immunoprecipitation assay, 293 T cells were transfectedwith MyoD for 36 h. After C2C12 or MyoD-transfected 293 T cellswere treated with EC for 24 h, whole cell extracts were incubatedwith anti-E2A and protein G agarose beads overnight at 4 �C. Beadswere washed three times and resuspended in extraction buffer,and samples were analyzed by western blotting.

2.5. Immunocytochemistry and microscopy

Immunostaining for MHC expression was performed asdescribed previously (Bae et al., 2009). Briefly, C2C12 cells werefixed with 4% paraformaldehyde, permeabilized with 0.2% TritonX-100 in phosphate buffered saline, blocked, and stained withanti-MHC, followed by an Alexa Fluor 568-conjugated secondaryantibody (Molecular Probes, Eugene, OR). Images were capturedand processed with a Nikon ECLIPSE TE-2000U microscope andNIS-Elements F software (Nikon, Tokyo, Japan).

2.6. Luciferase assay

C2C12 cells were seeded in 12-well plates at a density of4 � 104 cells per well. Twenty-four hours after seeding, C2C12 cells

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were transfected with 100 ng of 4RTK-luciferase reporter by usingLipofectamin 2000. Twenty-four hours later, transfected cells weretreated with the indicated concentration of daidzein and differen-tiated in DM for 36 h followed by luciferase activity measurementwith Luciferase Reporter Assay System (Promega, Sunnyvale, CA).293 T cells were seeded in 12-well plates at a density of 4 � 104

cells per well for 24 h and were transfected with 100 ng of 4RTK-luciferase reporter and cotransfected with 50 ng of MyoD by usingLipofectamin 2000. Twenty-four hours later, transfected cells weretreated with the indicated concentration of daidzein and incubatedfor 24 h followed by luciferase activity measurement. Experimentswere performed in triplicates and repeated at least three timesindependently.

2.7. Statistics

The experiments were done independently three times. Theparticipants’ T-test was used to access the significance of the differ-ence between two mean values. ⁄P < 0.01 and ⁄⁄P < 0.05 were con-sidered to be statistically significant.

3. Results

3.1. Daidzein prevents myotube atrophy triggered by dexamethasonethrough Akt activation

It has been reported that the synthetic glucocorticoid dexam-ethasone (DEX) triggers myotube atrophy in vivo and in vitro(Hasselgren, 1999; Stitt et al., 2004). By utilizing DEX-treatedC2C12 myotubes, we have investigated whether daidzein protectsmyotubes from glucocorticoid-induced muscle atrophy. C2C12myoblasts were induced to differentiate in differentiation medium(DM) for 1 day (D1) followed by the treatment with 400 ng/ml DEXalong with vehicle or daidzein for additional 2 days (D3). Thesecells were then subjected to immunostaining for myosin heavychain (MHC) to assess myotube formation. Daidzein treatmentenhanced myotube formation as seen by a higher proportion of lar-ger myotubes containing more than six nuclei, relative to thevehicle-treated cells (Fig. 1A and B). As expected, DEX-treatmentblocked myotube formation and the majority of cells remained asmononucleated myocytes, whereas daidzein treatment in DEX-treated cells restored myotube formation, as evident by the pres-ence of larger multinucleated myotubes with more than six nuclei(Fig. 1A and B). The quantification of myotube diameter revealedthat DEX treatment evoked a decrease in myotube diameter, whichwas partially restored by daidzein (Fig. 1C).

In the replica experiments, the expression levels of muscle-specific proteins (MHC, MyoD and Myogenin) and key promyo-genic kinases (p38MAPK and Akt) were determined. As shown inFig. 1D, daidzein treatment elevated MHC, MyoD and Myogeninlevels, while DEX treatment reduced the expression of these pro-teins (Fig. 1D). In agreement with the immunoblotting data shownin Fig. 1A, the rescue effect of daidzein treatment on DEX-inducedatrophic myotubes was also reflected by the restored expression ofMHC and Myogenin, similar to the level of vehicle control (Fig. 1D).In addition, the treatment of daidzein resulted in dramaticallyincreased levels the active phosphorylated form of p38MAPK(pp38) and Akt (pAkt) (Fig. 1E). Control DEX-treated cells haddecreased levels of pp38 and pAkt, while daidzein treatment par-tially restored the levels of pAkt in DEX-induced atrophic myo-tubes, similarly to those of control myotubes. However daidzeintreatment did not alter pp38 levels in these cells. These results sug-gest that daidzein prevents myotube atrophy triggered by DEXthrough Akt activation.

3.2. Black soybean extract and daidzein promote myoblastdifferentiation

To further investigate this beneficial effect of daidzein on myo-tube, we determined the effect of black soybean (Glycine max (L.)Merr) extract and daidzein on myoblast differentiation and myo-tube hypertrophy. The isoflavonoid profile of BS Ext representedin Fig. 2A. To examine the effect of BS Ext in myoblast differentia-tion, C2C12 myoblasts were induced to differentiate for 2 days (D2)with the addition of vehicle DMSO or the indicated amount of BSExt to the differentiation medium and assessed for the effect onmyoblast differentiation by immunoblotting for the expression ofmuscle-specific proteins. The treatment with BS extract increasedthe expression of MHC, MyoD and Myogenin proteins in a dose-dependent manner (Fig. 2B). As assessed by MHC immunostaining,C2C12 myoblasts treated with BS Ext for 2 days formed biggerMHC-positive myotubes with more nuclei compared to the controltreated cells (Fig. 2C). To quantify myotube formation, MHC-positive cells were counted as mononucleate, containing two tofive nuclei, or containing six or more nuclei per myotube and plot-ted as percentile (Fig. 2D). The treatment with BS Ext reduced theproportion of mononucleate myocytes, while it substantially ele-vated the proportion of bigger myotubes containing six or morenuclei in a dose-dependent manner (Fig. 2D).

To further confirm the promyogenic effect of BS Ext, we haveexamined the effect of daidzein, one of representative isoflavonesfrom black soybean, on myoblast differentiation, C2C12 myoblastswere induced to differentiate for 2 days in presence of vehicleDMSO or the indicated concentration of daidzein ranging from 1to 50 nM followed by myoblast differentiation analysis. Consistentwith the result of BS Ext, C2C12myoblasts treated with daidzein for2 days formed a higher proportion of larger MHC-positive myo-tubes, compared to control-treated cells (Fig. 2E). To quantify theextent of myotube formation, MHC-positive cells were scored asmononucleate, containing two to five nuclei, or containing six ormore nuclei per myotubes and plotted as percentile (Fig. 2F). Inthe daidzein treated culture, the proportion of mononucleate cellswas decreased while the percentile of large myotubes containingsix or more nuclei proportionally increased in a dose-dependentmanner (Fig. 2F). Furthermore, daidzein treatment elevated MHC,MyoD and Myogenin levels in a dose-dependent manner, as as-sessed by immunoblotting (Fig. 2G). Collectively, these data demon-strate that BS Ext and daidzein promote myoblast differentiation.

3.3. The treatment of BS extract and daidzein promote myoblastdifferentiation through activation of p38MAPK and Akt

To assess the molecular regulatory mechanisms of BS Ext ordaidzein-elicited myoblast differentiation, C2C12 myoblasts weretreated with varying concentration of BS Ext or daidzein andassessed for the activation status of promyogenic kinases,p38MAPK and Akt. The treatment of BS Ext enhanced pp38 andpAkt levels in a dose-dependent manner, whereas the level of totalproteins stayed relatively constant (Fig. 3A). Especially, the effec-tive concentration of BS Ext to activate p38MAPK in C2C12 myo-blasts ranged from 1 to 10 ng/mL. Similarly to the resultsobtained with BS Ext treatment, the treatment of daidzein dramat-ically increased pp38 and pAkt levels in a dose-dependent manner,without changes in total protein levels (Fig. 3B). Next we examinedwhether the activation of p38MAPK and Akt is required for thedaidzein-elicited promyogenic effect. To do this, C2C12 myoblastswere pretreated with pharmacological inhibitors for p38MAPK(SB203580) or Akt (LY294002) for 30 min, respectively, prior tothe daidzein treatment for 48 h in DM. Then, the expression ofpp38, pAkt, MHC, MyoD and Myogenin was assessed. Inhibitionof either Akt by LY294002 or p38MAPK by SB203580 resulted in

Fig. 1. Daidzein prevented DEX-induced myotube atrophy through activation of Akt signaling. (A) C2C12 myoblasts were induced to differentiate in DM for 1 day and thentreated with 400 ng/mL DEX along with vehicle or 50 nM daidzein in fresh DM for the additional 2 days, followed by immunostained for MHC expression (red) and DAPI tovisualize nuclei (blue) to reveal myotube formation. (B) Quantification of myotube formation from data shown in panel (A). Value represent means of triplicatedeterminations ± 1 SD. The experiment was repeated three times with similar results. Asterisks indicate significant difference from the control. *P < 0.01, **P < 0.05. (C)Quantification of average myotube diameter from data shown in panel (A). Values represent means of determination of 6 fields ± 1 SD. The experiment was repeated withsimilar results. Asterisks indicate significant difference from the control. *P < 0.01, **P < 0.05. (D) Cell lysates indicated in panel (A) were subjected to immunoblotting withantibodies to MHC, MyoDMyogenin and pan-Cadherin as a loading control. The experiment was repeated three times with similar results. (E) Cell lysates from panel (A) weresubjected to immunoblotting with antibodies to pAkt, pp38, Akt and p38 as loading controls, respectively. The experiment was repeated three times with similar results. (Forinterpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 2. Daidzein enhances myotube formation of C2C12 myoblasts. (A) UV (254 nm) chromatogram of BS Ext. Compounds 1–7 are highlighted as shown above; daidzin (1),glycitin (2), genistin (3), malonyldaidzin (4), malnylglycitin (5), malonylgenistin (6), and daidzein (7). (B) C2C12 myoblasts were treated with indicated concentration of BSExt and differentiated in DM for 2 days, followed by immunoblotting with antibodies to MHC, MyoD, Myogenin and pan-Cadherin as a loading control. The experiment wasrepeated three times with similar results. (C) C2C12 myoblasts from similar experiments shown in panel (B) were subjected to immunostaining for MHC expression (red) andDAPI to visualize nuclei (blue) to reveal myotube formation. (D) Quantification of myotube formation from data shown in panel (C). Value represent means of triplicatedeterminations ± 1 SD. The experiment was repeated three times with similar results. Asterisks indicate significant difference from the control. *P < 0.01. (E) C2C12 myoblastswere treated with indicated concentration of daidzein and differentiated in DM for 2 days, followed by immunostained for MHC expression (red) and DAPI to visualize nuclei(blue) to reveal myotube formation. (F) Quantification of myotube formation from data shown in panel (E). Value represent means of triplicate determinations ± 1 SD. Theexperiment was repeated three times with similar results. Asterisks indicate significant difference from the control. *P < 0.01. (G) Cell lysates from similar experiments shownin panel (E) were subjected to immunoblotting with antibodies to MHC, MyoD, Myogenin and pan-Cadherin as a loading control. The experiment was repeated three timeswith similar results. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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diminished myoblast differentiation, evident by blunted expres-sion of MHC, MyoD and Myogenin, compared to the vehicle-treated cells (Fig. 3C and D). Daidzein treatment failed to restorethe expression of MHC, MyoD and Myogenin in Akt- orp38MAPK-inhibited cells. These results suggest that the promyo-genic effect of daidzein is dependent on activation of p38MAPKand Akt pathways to enhance MyoD activities and myoblastdifferentiation.

3.4. Daidzein enhances MyoD activity and heterodimerization of MyoDwith E proteins

To further examine the effect of daidzein on MyoD activation,293T cells were cotransfected with a MyoD-responsive luciferasereporter and the expression vector for MyoD. Twenty-four hourslater, lysates were subjected to luciferase assay. The treatment ofdaidzein increased the MyoD-responsive luciferase activity in adose-dependent manner and especially 10 and 50 nM daidzeinenhanced the luciferase activity about 1.9-fold and 3.2-fold,

respectively (Fig. 4A). To confirm the daidzein effect on endoge-nous MyoD activity, C2C12 myoblasts were transfected with aMyoD-responsive luciferase reporter and 24 h later, myoblastswere induced to differentiate in presence of vehicle or the indi-cated concentration of daidzein for additional 36 h, followed byluciferase assay. As shown in Fig. 4B, daidzein increased theMyoD-dependent luciferase activities in a dose-dependent mannerand enhanced the luciferase activity approximately 3.2-fold. Thesedata suggest that daidzein enhances MyoD activities.

One of major regulatory mechanism for MyoD activation is itsheterodimerization with E proteins. p38MAPK and Akt augmentMyoD activation through enhancing its heterodimerization (Baeet al., 2009; Wilson & Rotwein, 2007). Therefore we examined theeffect of daidzein on heterodimer formation of MyoD with E2A.To do this, 293T cells expressing MyoD were treated with vehicleor daidzein for two days, followed by immunoprecipitation withantibodies to E2A and immunoblotting with MyoD antibodies.The E2A protein level in total lysates was unaltered regardless ofthe daidzein treatment, while MyoD expression was substantially

Fig. 3. Daidzein induces activation of Akt and p38MAPK dose-dependently. C2C12 myoblasts were treated with (A) BS Ext or (B) daidzein and induced to differentiate in DMfor 2 days. Cell lysates were subjected to immunoblotting with antibodies to pAkt, Akt, pp38 and p38. The experiment was repeated three times with similar results. (C)C2C12 myoblasts were treated with 1 lM LY294002 for 30 min prior to the treatment with daidzein, and then differentiated in DM for 2 days. Cell lysates were subjected toimmunoblotting with antibodies to pAkt, Akt, MHC, MyoD, Myogenin and pan-Cadherin as a loading control. (D) C2C12 myoblasts were treated with 2.5 lM SB203580 for30 min prior to the treatment with daidzein, and then differentiated in DM for 2 days. Cell lysates were subjected to immunoblotting with antibodies to pp38, p38, MHC,MyoD, Myogenin and pan-Cadherin as a loading control.

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increased in daidzein-treated cells, relatively to the vehicle-treatedcells (Fig. 4C). Furthermore, the amount of MyoD in precipitateswith the E2A antibody was greatly elevated in MyoD-transfected293T cells, compared to the vehicle-treated cells (Fig. 4C). To fur-ther examine, C2C12 myoblasts were treated with vehicle or daid-zein for two days in DM, followed by co-immunoprecipitationanalysis. Consistent with previous results, more MyoD proteinswere found in the precipitates with E2A antibodies in daidzein-treated C2C12 myoblasts, compared to the control cells (Fig. 4D).Consistently, the level of MyoD increased mildly in daidzein-treated C2C12 myoblasts, relative to the vehicle-treated cells(Fig. 4D). These data suggest that daidzein enhances MyoD activityby augmenting its heterodimerization with E proteins, likelythrough p38MAPK and Akt.

3.5. Daidzein evokes myotube hypertrophy via Akt/mTOR/p70S6kinase pathway

Constitutive activation of Akt in muscle has been implicated inmuscle hypertrophy through activation of mammalian target ofrapamycin (mTOR) and p70S6 kinase (Bodine et al., 2001). Toinvestigate the effect of BS Ext on muscle hypertrophy, C2C12myoblasts were induced to differentiate for 2 days (D2), followedby the treatment with 100 ng/mL BS Ext for additional 2 days(D4). As shown in Fig. 5A, myotubes treated with BS Ext has higherlevels of MHC, MyoD and Myogenin proteins, compared to controlmyotubes. The treatment with BS Ext increased the activation ofAkt and the levels of phosphorylated forms of its downstreamhypertrophy signals including mTOR and p70S6K (Fig. 5B).

To further define whether daidzein promotes Akt-mediatedmyotube hypertrophy, 50 nM daidzein treatment at late stage ofC2C12 myoblast differentiation induced myotube formation andsubjected to immunostaining for MHC to assess myotube forma-tion. Daidzein-treated C2C12 cells formed more of larger myo-tubes, relative to the control-treated cultures (Fig. 5C). Themeasurement of myotube diameters revealed that daidzein treat-ment enhanced the myotube thickness about 2.3-fold (Fig. 5D).To further examine the effect of daidzein on expression ofmuscle-specific proteins and involved signaling pathways, C2C12myoblasts were cultured under the same experimental conditionsas mentioned above, subjected to immunoblotting analysis. Simi-larly to early differentiation, myotubes treated with daidzein dis-played higher levels of MHC, MyoD and Myogenin expression,compared to control myotubes (Fig. 5E). Daidzein treatment dra-matically promoted activation of Akt and Akt downstream targets,as evident by greatly enhanced levels of the phosphorylated formsof mTOR and p70S6K, compared to vehicle-treated cells (Fig. 5F).However, under this condition, daidzein treatment did not alterthe level of pp38. These results suggest that daidzein elicits activa-tion of Akt signaling pathway to induce myotube hypertrophy.

3.6. Daidzein promotes a myogenic conversion of fibroblasts mediatedby MyoD

MyoD as a master myogenic factor can convert fibroblasts intomyogenic lineage (Tapscott et al., 1988). Since daidzein augmentsMyoD activity, we investigated its effect on the myogenic conver-sion of fibroblasts. 10T1/2 mouse embryonic fibroblasts expressing

Fig. 4. Daidzein treatment enhances MyoD activities and heterodimerization of MyoD with E proteins. (A) 293T cells were transfected with 4RTK-luciferase reporter andcotransfected with MyoD. Twenty-four hours later, 293 T cells were treated with indicated concentration of daidzein and incubated for 24 h, followed by luciferase assay.Asterisks indicate significant difference from the control at *P < 0.01, **P < 0.05. (B) C2C12 myoblasts were transiently transfected with 4RTK-luciferases reporter. After 24 h,C2C12 myoblasts were treated with indicated concentration of daidzein and induced to differentiate for 36 h, followed by luciferase assay. Values are means of triplicatedeterminants. Asterisks indicate significant difference from the control at *P < 0.01. (C) MyoD-transfected 293 T cells and (D) C2C12 myoblasts were treated with DMOS ordaidzein and subjected to immunoprecipitation with E2A antibodies followed by immunoblotting analysis with MyoD antibodies. Total lysates are shown as an input controlfor each protein. The experiment was repeated three times with similar results. The relative MyoD intensity of daidzein to vehicle was quantified and added under the blot.

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MyoDwere treatedwith DMSO or daidzein and induced to differen-tiation for two days, followed by immunoblot analysis. The vehicle-treated 10T1/2/MyoD cells induced mildly MyoD, Myogenin andMHC expression, which were further enhanced by daidzein treat-ment (Fig. 6A). In addition, we investigated the activation statusof Akt and p38 in 10T1/2 cells. Consistently, daidzein treatmentenhanced the levels of pAkt and pp38 (Fig. 6B). Taken together,these data demonstrate that daidzein enhances the MyoD-dependent myogenic conversion of fibroblasts via Akt and p38.

4. Discussion

Much effort has been paid to identify effective nutritional andpharmacological supplements to improve muscle function and toreinforce the muscle strength in pathological conditions or agingprocess. Previously, it has been reported that daidzein preventsMuRF1-mediated muscular atrophy through SIRT1 activation(Hirasaka et al., 2013). In current study, we investigate the agonis-tic effects and molecular mechanisms of black soybean extract anddaidzein as potential compounds for promoting muscle differenti-ation and hypertrophy. Our data demonstrate that black soybeanextract and daidzein, one of representative isoflavones from blacksoybean elicit myoblast differentiation through activation of Aktand p38MAPK which in turn up-regulates MyoD-dependent myo-

genic differentiation. In addition, daidzein protects myotube atro-phy triggered by DEX and induces myotube hypertrophy throughactivation of Akt, mTOR and p70S6K pathway. Akt and p38MAPKplay key roles in myogenic differentiation through activation ofMyoD which in turn induces muscle-specific gene expressionincluding MHC and Myogenin (Bae et al., 2010; Bergstrom et al.,2002; Kaneko et al., 2002) and these myogenic signalings areinvolved in a positive feedback network that amplifies and main-tains the myogenic phenotype (Ludolph & Konieczny, 1995).

Phytoestrogens are plant phenolic compounds that are similarin structure to mammalian estrogens and exhibit biological func-tions comparable to estrogens (Patisaul & Jefferson, 2010). In thegut, phytoestrogens are further metabolized by microflora intomore active compounds such as equol or O-Desmethylangolensin(Heinonen, Wahala, & Adlercreutz, 1999). After ingestion, daidzeinis converted to equol by the gut microflora that has been known asa soybean isoflavone metabolite (Rowland, Wiseman, Sanders,Adlercreutz, & Bowey, 2000). Black soybean increases GLUT4translocation and glucose transport in myotubes and restores thesuppression of insulin-mediated Akt phosphorylation in insulin-resistant cells (Jang et al., 2010). Equol, a daidzein derivative, hasbeen reported to promote glucose uptake, AMPK phosphorylationand GLUT4 translocation in L6 myotubes in absence of insulinand reduces fasting blood glucose levels and gene expression ofhepatic enzymes related to glucose metabolism (Cheong et al.,

Fig. 5. Daidzein promotes Akt-dependent myotube hypertrophy. (A) C2C12 myoblasts were induced to differentiate in DM for 2 days and then treated with 100 ng/mL BS Extfor the additional 2 days. Cell lysates were subjected to immunoblotting with antibodies to MHC, MyoDMyogenin and pan-Cadherin as a loading control. The experiment wasrepeated three times with similar results. (B) Cell lysates from panel (A) were subjected to immunoblotting with antibodies to pAkt, Akt, p-mTOR, mTOR, p-p70S6K, p70S6K,p-4E-BP1, 4E-BP1. The experiment was repeated three times with similar results. (C) C2C12 myoblasts were induced to differentiate in DM for 2 days and then treated with50 nM daidzein for the additional 2 days. Cells were immunostained for MHC expression (red) and DAPI to visualize nuclei (blue) to reveal myotube formation. (D)Quantification of average myotube diameter from data shown in panel (C). Values represent means of determination of 6 fields ± 1 SD. The experiment was repeated withsimilar results. Asterisks indicate significant difference from the control. *P < 0.01. (E) Cell lysates indicated in panel (C) were subjected to immunoblotting with antibodies toMHC, MyoD Myogenin and pan-Cadherin as a loading control. The experiment was repeated three times with similar results. (F) Cell lysates from panel (C) were subjected toimmunoblotting with antibodies to pAkt, Akt, p-mTOR, mTOR, p-p70S6K, p70S6K, pp38, p38, a-tubulin as loading controls. The experiment was repeated three times withsimilar results. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6. Daidzein augments myogenic differentiation of MyoD-transfected 10T1/2embryonic fibroblasts. (A) 10T1/2 cells were transiently transfected with MyoDexpression vector (pBp-MyoD) and induced to differentiate with the treatment ofeither 50 nM daidzein or DMSO for 2 days, respectively. Cell lysates wereimmunoblotted with antibodies to MHC, MyoD, Myogenin and pan-Cadherin as aloading control. (B) Cell lysates shown in panel (A) were subjected to immunoblot-ting with antibodies to pAkt, Akt, pp38 and p38.

S.-J. Lee et al. / Journal of Functional Foods 38 (2017) 438–446 445

2014a). Daidzein promotes glucose uptake through GLUT4 translo-cation to plasma membrane in L6 myocytes and improves glucosehomeostasis in type 2 diabetes model mice (Cheong et al., 2014b).Previously, we reported that Syntaxin 4 (Stx4) promotes myoblast

differentiation through interaction with a promyogenic receptorCdo and stimulation of its surface translocation (Yoo et al., 2015).Both Cdo and Stx4 are required for GLUT4 translocation to cell sur-face and glucose uptake in myoblast differentiation. Furthermore,Cdo signals through Akt and p38MAPK, thereby enhancing MyoDactivities and myoblast differentiation (Bae et al., 2009, 2010;Takaesu et al., 2006). Considering that daidzein exerts promyo-genic effects through resembling mechanisms of native promyo-genic signaling, daidzein might be an effective compound toenhance endogenous muscle stem cell differentiation.

The Akt/mTOR pathway plays a major role in muscle hypertro-phy through regulating protein synthesis leading to muscle growth(Bodine et al., 2001; Stitt et al., 2004). Previously, it has shown thatgenetic activation or ablation of Akt/mTOR pathway induced mus-cle hypertrophy through prevention of atrophic response or inhib-ited hypertrophy, respectively (Bodine et al., 2001). In line withthis notion, daidzein treatment at late stage of myoblast differenti-ation induced hypertrophic myotube formation and daidzein treat-ment prevented myotube atrophy triggered by dexamethasone.Interestingly, daidzein activates Akt/mTOR pathway to inducehypertrophic responses in the late stages of myogenic differentia-tion or dexamethasone-triggered myotube atrophy. The improve-ment of muscle stem cell function is of importance to treataging- or disease-associated muscle atrophy, thus daidzein mightbe an effective nutritional supplement for the treatment ofaging-related or pathologically muscle loss and weakness.

446 S.-J. Lee et al. / Journal of Functional Foods 38 (2017) 438–446

5. Conclusions

Our study demonstrates that daidzein promotes myogenic dif-ferentiation and myotube growth through activation of Akt andp38 that in turn stimulates MyoD-mediated muscle-specific geneexpression. Furthermore, daidzein increments myogenic conver-sion of embryonic fibroblasts and myogenic differentiation. Thusdaidzein has a great potential as a therapeutic or neutraceuticalremedy to intervene muscle weakness and atrophy.

Acknowledgements

Thisworkwas supported by theNational Research Foundation ofKorea (NRF) grant funded by the Korea government (MSIP) (NRF-2011-0030074, 2015R1A2A1A15056117, 2016R1A2B4014868).

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