Glabridin inhibits dexamethasone-induced muscle atrophy

Accepted Manuscript Glabridin inhibits dexamethasone-induced muscle atrophy Yasukiyo Yoshioka, Yusuke Kubota, Yumi Samukawa, Yoko Yamashita, Hitoshi Ashida PII:

S0003-9861(18)30873-7

DOI:

https://doi.org/10.1016/j.abb.2019.02.006

Reference:

YABBI 7951

To appear in:

Archives of Biochemistry and Biophysics

Received Date: 23 October 2018 Revised Date:

7 February 2019

Accepted Date: 12 February 2019

Please cite this article as: Y. Yoshioka, Y. Kubota, Y. Samukawa, Y. Yamashita, H. Ashida, Glabridin inhibits dexamethasone-induced muscle atrophy, Archives of Biochemistry and Biophysics (2019), doi: https://doi.org/10.1016/j.abb.2019.02.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Title

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Glabridin inhibits dexamethasone-induced muscle atrophy

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Authors

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Yasukiyo Yoshiokaa, Yusuke Kubotab, Yumi Samukawab, Yoko Yamashitab, Hitoshi

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Ashidab*

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Affiliations

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a

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Hyogo 657-8501, Japan

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b

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Japan

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Graduate School of Science, Technology and Innovation, Kobe University, Kobe,

Graduate School of Agricultural Science, Kobe University, Kobe, Hyogo 651-8501,

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*Corresponding author

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Hitoshi Ashida

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Postal address: Graduate School of Agricultural Science, Kobe University, 1-1,

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Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan. Tel: +81-78-803-5878.

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E-mail: [email protected].

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Abstract

Prevention of muscle wasting is known to contribute to improving the quality

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of life and extending a healthy life. Recently, we have reported that licorice flavonoid

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oil containing glabridin, which is a prenylated isoflavone, enhances muscle mass in

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mice. In this study, we investigated the prevention effect of glabridin on

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dexamethasone-induced muscle atrophy and clarified its mechanism in cultured

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myotubes and in muscle of mice. Treatment with glabridin to C2C12 myotubes 1

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inhibited dexamethasone-induced protein degradation through dexamethasone-induced

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expression of ubiquitin ligases, MuRF1 and Cbl-b, but not atrogin-1. Mechanistically,

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glabridin inhibited nuclear translocation of the glucocorticoid receptor. Glabridin

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directly bound to the glucocorticoid receptor, resulting in the inhibition of binding

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between dexamethasone and the receptor protein. Glabridin also inhibited

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dexamethasone-induced phosphorylation of p38 and FoxO3a, as the upstream for the

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induction of ubiquitin ligases in C2C12 myotubes. Moreover, the glabridin-induced

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inhibition of protein degradation was eliminated by knockdown of the glucocorticoid

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receptor, but not by p38 knockdown. These data indicated that the inhibitory

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mechanism of glabridin against dexamethasone-induced muscle atrophy was mainly

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mediated by the inhibition of binding between dexamethasone and the glucocorticoid

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receptor

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dexamethasone-induced protein degradation in the tibialis anterior muscle of mice. It

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was confirmed that glabridin inhibited dexamethasone-induced nuclear translocation of

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the glucocorticoid receptor and phosphorylation of FoxO3a in the muscle of mice.

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These findings suggest that glabridin is an effective food ingredient for the prevention

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of glucocorticoid-induced skeletal muscle atrophy.

myotubes.

Oral

administration

of

glabridin

prevented

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Glabridin inhibited binding of dexamethasone to the glucocorticoid receptor

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Administration of glabridin eliminated dexamethasone-induced muscle atrophy

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Highlights •

Glabridin inhibited dexamethasone-induced protein degradation in C2C12

myotubes

in mice

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Key words

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muscle atrophy; glucocorticoid receptor; glabridin

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Abbreviations used

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MuRF1; muscle ring finger 1, FoxO; fork head box O, DMEM; Dulbecco’s modified

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Eagle's minimal essential medium, FBS; fetal bovine serum, PCA; perchloric acid,

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Introduction

Skeletal muscle mass is maintained by a balance between synthesis and

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degradation of muscle protein, and it is known that the collapse of this balance causes

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muscle wasting [1]. Because muscle atrophy, which is characterized by muscle

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wasting, causes deterioration of the quality of life and shortening of a healthy life, its

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prevention is one of the important social issues in recent years. It is generally

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considered that only rehabilitation is effective for muscle atrophy. However, this is not

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a realistic treatment plan because patients with this disease are not very mobile.

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Therefore, prevention of muscle atrophy by ingestion of functional foods is an

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attractive approach. Previous reports demonstrated that certain food ingredients

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prevented muscle atrophy: For example, epigallocatechin gallate and β-carotene

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prevented muscle atrophy through their antioxidant activity in rodents [2,3]. However,

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food ingredients that specifically modulate synthesis or degradation of muscle protein

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have not been reported sufficiently. Therefore, it is important to investigate search for

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the effective food factors that can modulate muscle mass through induction of

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hypertrophy or inhibition of atrophy.

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Skeletal muscle atrophy characterized by protein degradation is often caused

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by multiple pathophysiologic conditions. Glucocorticoid, an endocrine hormone 3

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secreted from the adrenal cortex, is involved in various types of muscle atrophy

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including sarcopenia [4]. Glucocorticoid is an important mediator for muscle protein

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degradation and upregulation of the ubiquitin proteasome pathway in skeletal muscle

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[5]. Treatment of C2C12 myotubes with dexamethasone, a synthesized glucocorticoid

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analogue, is often used for in vivo and in vitro muscle atrophy models [6,7].

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The ubiquitin proteasome pathway is mainly involved in protein degradation

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in skeletal muscle [8]. Ubiquitin ligase plays a major role in muscle atrophy and its

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activation directly influences muscle mass to decrease [9]. Atrogin-1, muscle ring

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finger 1 (MuRF1), and Cbl-b are known as myocardial and skeletal muscle-specific

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ubiquitin ligases, which are used as the markers for muscle atrophy [10,11].

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Transcription factor of fork head box O (FoxO) is known as a master regulator of these

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ubiquitin ligases [12]. It has been reported that dexamethasone-induced myotube

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atrophy is accompanied by upregulation of ubiquitin ligases and activation of FoxO1

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and FoxO3 [13]. p38 Mitogen-activated protein kinase activates FoxO1 and FoxO3a

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[14].

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Licorice, the root of Glycyrrhiza glabra L., is frequently used as traditional

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herbal medicine and food in many countries. Licorice has been reported to possess

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antidiabetic, anti-inflammatory, antimicrobial, antioxidant, and antitumor activities

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[15-22]. Glabridin, a prenylated isoflavone, is a major compound in licorice and also

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possesses various health beneficial effects, such as anti-diabetes [23], anti-oxidative

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[24], ant-tumor [25], anti-inflammatory [26], and anti-obesity [27]. Recently, we have

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reported that licorice flavonoid oil containing glabridin inhibits muscle atrophy in

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KK-Ay mice [28]. It is, however, unclear whether glabridin itself inhibits muscle

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atrophy or not. Therefore, in this study, we investigated the prevention effect of

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glabridin on dexamethasone-induced muscle atrophy and its molecular mechanism.

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Materials & Methods

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Reagents Glabridin and dexamethasone was purchased from Wako Pure Chemical

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Industries (Osaka, Japan). Antibodies against phosphorylated FoxO3a (Ser7), FoxO3a,

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phosphorylated FoxO1 (Ser256), FoxO1, phosphorylated p38 (Ser180/Tyr182), p38,

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glucocorticoid receptor and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

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were purchased from Cell Signaling Technology Co. (Denvers, MA, USA). Antibodies

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against MuRF1 and atrogin-1 were from Abcam (Cambridge, UK). Horseradish

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peroxidase (HRP)-conjugated anti-mouse IgG, HRP-conjugated anti-goat IgG

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antibodies, Lamin B and Cbl-b were from Santa Cruz Biotechnology (Santa Cruz, CA,

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USA). HRP-conjugated anti-rabbit IgG was from Bio-Rad Laboratories Inc. (Hercules,

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CA, USA). Bovine serum albumin, Blocking One, and Blocking One-P solutions were

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from Nacalai Tesque, Inc. (Kyoto, Japan). Polyvinylidene difluoride membrane was

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obtained

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chemiluminescence detection kits were products of Wako Pure Chemical Industries.

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L-[ring-3,

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from Perkin Elmer Inc. (Boston, MA, USA). Human recombinant glucocorticoid

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receptor was from Life Technologies Japan (Tokyo, Japan). FG linker beads were from

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Tamagawa Seiki (Nagano, Japan). All other reagents used were of the highest grade

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available from commercial sources.

GE

Healthcare

(Fairfield,

WA,

USA).

Immunostar®

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5-3H]-Tyrosine and [1, 2, 4, 6, 7-3H (N)]-dexamethasone were purchased

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Cell culture Mouse skeletal muscle C2C12 cells were plated on the dish coated with cell matrix type C (Nitta Gelatin, Osaka, Japan) and maintained in Dulbecco’s modified 5

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Eagle's minimal essential medium (DMEM) supplemented with 10% (v/v) fetal bovine

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serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM L–glutamine at

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37°C in a 95% (v/v) air-5% (v/v) CO2 atmosphere. For differentiation to myotubes,

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C2C12 cells were incubated for 5 days in DMEM containing with 2% (v/v) horse

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serum. For RNA interference, the target sequences for the glucocorticoid receptor, p38

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and negative control siRNA duplexes were as follows; siGR;

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CCAUUUCUGUUCAUGGCGCGU, sip38;

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UCAUGCUGAAUUGGAUGCACUAUAA and siCont;

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AAGUAACACUUGGCUAUUUCUTT. These duplexes were introduced into C2C12

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myotubes using Lipofectamine® RNAiMAX reagent (Thermo Fisher Scientific) for 48

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h according to the manufacturer’s instructions.

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Measurement of protein degradation in myotubes

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The rate of protein degradation was determined by measuring the release of PCA-soluble radioactivity from proteins pre-labeled with L–3H–tyrosine [29,30].

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Differentiated C2C12 myotubes were labeled with 0.04 MBq/mL of L–3H-tyrosine for

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24 h in DMEM containing 2% horse serum. The cells were rapidly rinsed six times

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with ice-cold PBS–tyrosine and then treated with several concentrations of glabridin

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for 30 min in DMEM containing 2 mM un-radioactive tyrosine. After incubation, 1.0

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µM dexamethasone was added and incubated for 24 h. The cultured medium was

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collected and 10 mg/mL bovine serum albumin and 10% (w/v) perchloric acid (PCA)

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were added and the mixture was incubated at 4°C for 1 h, followed by centrifugation at

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20,000 × g for 5 min at 4°C. The supernatant was used for determination of the

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PCA-soluble radioactivity in the medium. The protein precipitate was dissolved in 0.05

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M NaOH containing 0.1% (v/v) Triton-X-100. The cells were washed with ice-cold

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PBS and solubilized with 0.05 M NaOH containing 0.1% (v/v) Triton X-100. The

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PCA-soluble and PCA-insoluble radioactivity in the medium and the radioactivity in

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the cells were measured with Ultima Gold XR (Perkin Elmer) using a Tri-Carb 3110

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TR liquid-scintillation spectrometer (Perkin Elmer). Protein degradation was expressed

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as the ratio of protein degraded over the 24 h period and calculated as 100 times the

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PCA-soluble radioactivity in the medium divided by the sum of the PCA-soluble and

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PCA-insoluble radioactivity in the medium and radioactivity in the cells. Protein

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degradation was calculated from the following formula:

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Protein degradation (%) = (PCA-soluble radioactivity in the medium) ×100 /

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(PCA-soluble radioactivity in the medium + PCA-insoluble radioactivity in the

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medium + radioactivity in the cells)

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For determination of the intracellular protein levels, differentiated myotubes were incubated with 0.04 MBq/mL 3H-tyrosine for 24 h. After removing the

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supernatant, the cells were treated with several concentrations of glabridin for 30 min

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and stimulated with 1 µM dexamethasone for another 24 h. After stimulation, the cells

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were lysed with 0.05M NaOH containing 0.1% (v/v) Triton X-100 overnight at room

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temperature. The lysate was collected and 10 mg/mL bovine serum albumin and 10%

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(w/v) PCA were added and the mixture was incubated at 4°C for 1 h, followed by

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centrifugation at 20,000 × g for 10 min at 4 °C. The protein precipitate was dissolved

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in 0.05 M NaOH containing 0.1% (v/v) Triton X-100. The intracellular protein level

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was expressed as the ratio and calculated as 100 times the PCA-insoluble radioactivity

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in the cells divided by the amount of the other fraction of radioactivity. Intracellular

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protein levels were calculated from following formula:

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Intracellular protein levels (%) = (PCA-insoluble radioactivity in the cell) ×100/

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(PCA-insoluble radioactivity in the cell + PCA-soluble radioactivity in the cell + 7

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radioactivity in the medium)

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Cell viability assay C2C12 myotubes were incubated with 10% (v/v) FBS-DMEM for 24 h and

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treated with the defined concentrations of glabridin as noted in each figure for 24 h in a

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96 well plate. The cells were washed three times with PBS and stained with 0.2%

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(w/v) crystal violet for 10 min. After removing the excess stain with tap water, the cells

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were lysed with 50% (v/v) ethanol containing 0.5% (w/v) sodium dodecyl sulfate. The

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absorbance of the crystal violet in the lysate was measured at 530 nm using a Wallac 1420 ARVOsx (Perkin-Elmer).

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Preparation of the whole cell lysate and nuclear fraction

C2C12 myotubes were treated with several concentrations of glabridin for 30

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min and stimulated with 1.0 µM dexamethasone for 24 h in a 60 mm dish. After

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removing the supernatant, the cells were lysed with RIPA buffer, pH8.0 containing 50

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mM Tris, 150 mM sodium chloride, 1% (v/v) NP–40, 0.5% (w/v) deoxycholic acid,

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0.1% (w/v) sodium dodecyl sulfate, 0.5 mM dithiothreitol, 3 kinds of protease

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inhibitors (1 mM phenylmethylsulfonyl fluoride, 5 µg/mL leupeptin and 5 µg/mL

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aprotinin), and 2 kinds of phosphatase inhibitors [10 mM sodium fluoride and 1 mM

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sodium orthovanadate (V)]. The lysates were centrifuged at 20,000 × g for 20 min at

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4°C and the supernatants were used as the whole cell lysate. Preparation of the

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post-nuclear and nuclear fractions from the C2C12 myotubes was performed as

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previously described [31]. C2C12 myotubes were lysed with 20 mM HEPES buffer,

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pH7.6 containing 20% (v/v) glycerol, 10 mM sodium chloride, 1.5 mM magnesium

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chloride, 0.2 mM EDTA, 0.5 mM dithiothreitol, the protease inhibitors and

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phosphatase inhibitors. The lysate was centrifuged at 1,000 × g for 10 min at 4°C, and

2

obtained supernatant was used as a post-nuclear fraction. The precipitate was

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resuspended in hypertonic buffer consisting of 20 mM HEPES, pH 7.6, 20% (v/v)

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glycerol, 420 mM sodium chloride, 1.5 mM magnesium chloride, 0.2 mM EDTA, 0.5

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mM dithiothreitol, the protease inhibitors and phosphatase inhibitors. This suspension

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was incubated on ice for 1 h and centrifuged at 15,000 × g for 20 min at 4°C. The

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supernatant was used as a nuclear fraction.

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Western blot

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Lysate from the C2C12 myotubes and skeletal muscle was mixed with sodium dodecyl sulfate sample buffer, pH 6.8 containing 62.5 mM Tris, 2% (w/v) sodium

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dodecyl sulfate, 10% (v/v) glycerol, 5% (v/v) 2-mercaptoethanol, and 0.02% (w/v)

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bromophenol blue. The mixture was incubated at 100°C for 5 min and subjected to

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sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins in

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the gels were transferred onto a polyvinylidene fluoride membrane. The membrane

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was incubated with a blocking solution consisting of Blocking One (for detection of

17

unphosphorylated proteins) or Blocking one-P (for detection of phosphoproteins) for 1

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h at room temperature and sensitized with primary antibodies overnight at 4°C,

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followed by the corresponding horseradish peroxidase-conjugated secondary antibody

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for 1 h at room temperature. Protein bands were visualized using Immuno Star® LD

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Western Blotting Substrate and detected with Light-Capture II (ATTO, Tokyo, Japan).

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The density of the specific band was determined using ImageJ image analysis software

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(National Institutes of Health, Bethesda, MD, USA).

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Immunofluorescence staining 9

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C2C12 myotubes were cultured on cover glasses. Immunofluorescence staining was performed as previously described [32]. Briefly, the myotubes were fixed

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in 4% (w/v) paraformaldehyde and permeabilized with 0.1% (v/v) Triton X-100 and

4

then treated with blocking solution containing 10% (v/v) FBS, 5% (w/v) BSA, and

5

0.1% (w/v) sodium azide. The myotubes were sensitized with anti-GR primary

6

antibody in PBS containing 3% (w/v) BSA, followed by incubation with Alexa Fluor

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488-conjugated secondary anti-rabbit IgG antibody. The nucleus was stained with 10

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µg/mL 4’, 6-diamidino-2-phenylindole. The fluorescence level of Alexa Fluor 488 and

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4’, 6-diamidino-2-phenylindole was measured at 485/535 nm and 355/460 nm,

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respectively, using a FSX100 fluorescent microscope (OLYMPUS, Tokyo, Japan). For

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measurement of nuclear translocation, the numbers of glucocorticoid receptor located

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in the nuclei were counted (50–75 numbers of nuclei), and calculated as the percent of

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nuclear translocation.

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Competitive inhibition assay

Tibialis anterior muscle harvested from C57BL/6J mice was lysed with 10

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mM HEPES buffer, pH 7.9 containing 10 mM potassium chloride, 0.1 mM EDTA, 1

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mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5% (v/v) NP-40. The

19

muscle lysates were diluted with 20 mM HEPES buffer, pH 7.9 containing 100 mM

20

potassium chloride, 0.2 mM EDTA, 10% (v/v) glycerol, 1 mM dithiothreitol, and 0.2

21

mM phenylmethylsulfonyl fluoride to a final concentration of 0.1% NP-40. The lysates

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were sensitized with the anti-glucocorticoid receptor antibody overnight at 4°C. The

23

immune-complex solution was reacted with Protein A/G PLUS-Agarose (Santa Cruz

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Biotechnology) and rotated for 1 h and then centrifuged 200 × g for 5 min at 4°C. After

25

removing the supernatant, the precipitate was treated with 10 µM glabridin and rotated

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for 30 min at room temperature. The mixture was centrifuged 200 × g for 5 min and

2

treated with 2.6 MBq 3H-dexamethasone (Perkin Elmer) and rotated for 30 min at

3

room temperature. The immune-complex was centrifuged 200 × g for 5 min at 4°C.

4

After rinsing the precipitate, the radioactivity of the immune–complex was measured

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with Ulitima Gold XR (Perkin Elmer Inc.) using a Tri–Carb liquid-scintillation

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spectrometer (Perkin Elmer Inc.).

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Preparation of probes

Glabridin or the negative control daidzein were dissolved in N,

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N-dimethylformamide to create a 50 mM ligand solution. Each ligand solution was

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added to epoxy beads. The mixture was centrifuged at 20,000 × g for 5 min at room

12

temperature. After removing the supernatant, the pellet was resuspended in

13

dimethylformamide. The solution was dispersed using an ultrasonic device and

14

centrifuged again under the same conditions. After removing the supernatant, the

15

precipitate was resuspended in dimethylformamide and dispersed using an ultrasonic

16

devise. After centrifugation at 20,000 × g for 5 min, the precipitate was resuspended in

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dimethylformamide and added to the ligand solution. Potassium carbonate (at a ratio of

18

10 to 1 ligand) was added to the suspension and mixed using the ultrasonic device. The

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reaction was performed by incubation overnight at 60°C using a rotator. The incubated

20

solution was centrifuged at 20,000 × g for 5 min at room temperature. After the pellet

21

was washed twice with 50% N, N-dimethylformamide, the precipitate was suspended

22

in ultrapure water and dispersed using the ultrasonic device, and then centrifuged again

23

under the same conditions. The precipitate was washed three times with 50%

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methanol, suspended in 50% methanol, and then dispersed using an ultrasonic device

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under the same centrifugation conditions. The precipitate was resuspended in 50%

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methanol and stored at 4°C.

2 3

Binding assay The immobilized probes, glabridin or daidzein was mixed with 0.1 mg protein

5

of whole cell lysates from C2C12 myotubes or the recombinant glucocorticoid receptor

6

and incubated for 1 h at 4°C using the rotator. For the competition assay, glabridin was

7

mixed with 0.1 mg protein of the cell lysates or recombinant glucocorticoid receptor

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and incubated for 30 min at 4°C using the rotator. Then, the glabridin probe was added

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to the mixture and further incubated for 30 min at 4°C using the rotator. After the

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mixture was centrifuged at 20,000 × g for 5 min at 4°C, the supernatant was collected

11

as a flow through fraction (F). The precipitates were washed twice with 20 mM

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HEPES buffer, pH 7.9 containing 200 mM potassium chloride, 0.2 mM EDTA, 20%

13

(v/v) glycerol, 0.5% (v/v) NP-40, 1 mM dithiothreitol, and 0.2 mM

14

phenylmethylsulfonyl fluoride. The supernatant was collected as a wash fraction (W),

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while the precipitate was collected as an eluted fraction (E). These fractionations were

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subjected to western blotting for detection of the glucocorticoid receptor.

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Animal experiment

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The animal experiment was carried out according to the Guidelines for the

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Care and Use of Experimental Animals at Kobe University Rokkodai Campus. Male

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C57BL/6J mice (6 weeks old) were obtained from Japan SLC (Shizuoka, Japan) and

22

maintained in a temperature-controlled room (23 ± 2°C) with a 14:10 h light/dark cycle

23

(lights on at 8:00 a.m.). Mice were allowed free access to tap water and an AIN–93G

24

purified diet (Oriental Yeast Co. Ltd., Tokyo, Japan). Mice were divided into three

25

groups of 4 each: control, dexamethasone alone, and dexamethasone and glabridin. 12

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The mice were orally administrated glabridin (40 mg/kg body weight/day) or

2

polyethylene glycol as the vehicle control once a day for 2 weeks. After 1 week, mice

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were intraperitoneally injected with dexamethasone (5 mg/kg body weight/day) or

4

saline as the vehicle control once a day. At the end of experiment, the blood of the

5

mice was collected via cardiac puncture under anesthesia and then the mice were

6

sacrificed. The anterior, gastrocnemius, quadriceps, and soleus muscles were harvested

7

and their weights measured. The ratio of each skeletal muscle to the body weight was

8

calculated. Preparation of tissue lysate from the anterior muscle was performed as

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follows. The muscle was homogenized with 10-fold of RIPA buffer and placed on ice

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for 1 h. After centrifugation at 20,000 × g for 20 min at 4°C, the supernatant was

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collected and used as the tissue lysate. Preparation of the nuclear fraction from the

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anterior muscle was performed as described below. The muscle was homogenized in

13

20 mM HEPES lysis buffer, pH7.6 containing 20% (v/v) glycerol, 10 mM sodium

14

chloride, 1.5 mM magnesium chloride, 0.2 mM EDTA, 0.5 mM dithiothreitol, the

15

protease inhibitors and phosphatase inhibitors. The homogenate was centrifuged at

16

1,000 × g for 10 min at 4°C. The supernatant was used as the post-nuclear fraction.

17

The precipitate was resuspended in hypertonic buffer consisting of 20 mM HEPES, pH

18

7.6, 20% (v/v) glycerol, 420 mM sodium chloride, 1.5 mM magnesium chloride, 0.2

19

mM EDTA, 0.5 mM dithiothreitol, the protease inhibitors and phosphatase inhibitors.

20

The suspension was placed on ice for 1 h and centrifuged at 15,000 × g for 20 min at

21

4°C, and then the supernatant was used as the nuclear fraction. The tissue lysate,

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nuclear fraction, and post nuclear fraction from skeletal muscle were subjected to

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western blotting.

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Statistical analysis 13

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Statistical analysis was performed with JMP statistical software version 11.2.0 (SAS Institute. Cary, NC, USA). Data are represented as the means and standard error

3

(SE). The statistical significance of experimental observations was determined using

4

Tukey Kramer multiple comparison test and student’s t-test. The level of significance

5

was set as p < 0.05.

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Results

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Glabridin prevents dexamethasone-induced protein degradation in C2C12 myotubes

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We first examined whether glabridin inhibited dexamethasone-induced protein

10

degradation in C2C12 myotubes. Stimulation with 1 µM dexamethasone resulted in

11

protein degradation in C2C12 myotubes. Glabridin suppressed the radioactivity

12

released into the medium from pre-labeled protein in the cells. Treatment with

13

glabridin at 10 µM significantly inhibited dexamethasone-induced protein degradation

14

using the 3H-tyrosine-labeled system (Figure 1A). Next, the radioactivity remaining in

15

the intracellular protein was measured to confirm the effect of preventing protein

16

degradation.

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dexamethasone-decreased radioactivity in the intracellular protein, whereas this

18

compound did not affect protein degradation in the absence of dexamethasone (Figure

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1B). These results indicated that 10 µM glabridin significantly inhibited

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dexamethasone-induced protein degradation.

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significantly

canceled

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Glabridin prevents dexamethasone–induced expression of ubiquitin ligases in C2C12

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myotubes

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To

elucidate

the

preventive

mechanism

of

glabridin

against

dexamethasone-induced protein degradation, we focused on how the expression levels 14

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of MuRF1, Cbl-b, and atrogin-1 were related to muscle atrophy. As expected, treatment

2

with dexamethasone caused an increase in the expression level of MuRF1, Cbl-b, and

3

atrogin-1 in the C2C12 myotubes. Glabridin at 10 µM significantly inhibited

4

dexamethasone-induced expression of MuRF1 and Cbl-b in a dose-dependent manner,

5

but it failed to inhibit dexamethasone-induced expression of atrogin-1, (Figure 1C).

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Glabridin prevents translocation of glucocorticoid receptor to nuclei in C2C12

8

myotubes

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Treatment with dexamethasone caused translocation of the glucocorticoid

10

receptor to nuclei in the C2C12 myotubes (Figure 2A). Glabridin at 10 µM prevented

11

dexamethasone-induced translocation of the glucocorticoid receptor to nuclei.

12

Furthermore, glabridin did not affect the expression level of the glucocorticoid receptor

13

in the cell lysate (Figure 2A). To confirm these results, immunofluorescence

14

microscopy analysis was performed. It was observed that dexamethasone caused

15

nuclear translocation of the glucocorticoid receptor and treatment with glabridin

16

inhibited the action of dexamethasone as the same as the results from western blot

17

(Figure 2B).

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Glabridin inhibits binding of dexamethasone and the glucocorticoid receptor Since dexamethasone induced protein degradation through binding to the

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glucocorticoid receptor, we examined whether glabridin inhibited the binding of

22

dexamethasone to the glucocorticoid receptor. As a result, glabridin, but not daizein,

23

directly bound to the glucocorticoid receptor in myotubes (Figure 3A). Furthermore,

24

binding of the glabridin probe to the glucocorticoid receptor was canceled by

25

pre-treatment with glabridin (Figure 3B). Moreover, pre-treatment with glabridin 15

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inhibited the binding of 3H-dexamethasone to the glucocorticoid receptor in muscle

2

tissue (Figure 3C). These results indicate that glabridin inhibited the binding of

3

dexamethasone to the glucocorticoid receptor.

4 5 6

Glabridin prevents p38/FoxO3a signaling in C2C12 myotubes To

clarify

the

inhibitory

mechanism

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of

glabridin

against

dexamethasone-induced protein degradation, we examined whether glabridin inhibited

8

p38/FoxO3a signaling. Glabridin inhibited dexamethasone-induced phosphorylation of

9

FoxO3a, but not FoxO1 in C2C12 myotubes (Figure 4A). Moreover, dexamethasone

10

alone and combination of dexamethasone and glabridin did not affect phosphorylation

11

of mTOR, Akt at Ser473 and Thr308, and FoxO3a at Ser253 (Supplementary Figure

12

S1). Furthermore, the dexamethasone-induced phosphorylation of p38, an upstream

13

factor of FoxO3a, and FoxO3a was decreased by treatment with glabridin in the nuclei

14

(Figure 4B).

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Involvement of the expression of the glucocorticoid receptor and p38 in the inhibition

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of dexamethasone-induced protein degradation by glabridin

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Glabridin inhibited both the glucocorticoid receptor (Fig. 2) and p38/FoxO3

19

pathways (Fig. 4). To elucidate which pathway primarily contributed to the inhibition

20

of protein degradation, we introduced siRNA for the glucocorticoid receptor and p38.

21

Dexamethasone-induced

22

receptor-knocked down myotubes but not p38-knocked down myotubes (Figure 5).

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protein degradation

was canceled in

glucocorticoid

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Glabridin prevents dexamethasone-induced skeletal muscle atrophy in mice To confirm whether glabridin prevents dexamethasone-induced skeletal 16

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muscle atrophy in mice, glabridin (40 mg/kg body weight/day) was orally

2

administrated to C57BL/6J mice once a day for 2 weeks. After the first week, mice

3

were intraperitoneally injected with dexamethasone (5 mg/kg body weight/day) to

4

induce muscle atrophy. At the end of the experiment, muscle mass wasting was

5

observed in the tibialis anterior, gastrocnemius, and soleus muscle after the

6

intraperitoneal injection of dexamethasone alone (Table 1). Oral administration of

7

glabridin significantly suppressed the dexamethasone-decreased muscle weight of the

8

tibialis anterior muscle, and tended to suppress the decreased muscle weight of the

9

gastrocnemius muscle. When the total muscle mass was calculated, glabridin

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significantly suppressed the dexamethasone-induced muscle atrophy.

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13

Glabridin prevents dexamethasone-induced FoxO3a and GR signaling in mice To

confirm

the

inhibitory

mechanism

of

glabridin

against

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dexamethasone-induced skeletal muscle atrophy in mice, we examined the expression

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levels of MuRF1, Cbl-b and atrogin-1, phosphorylation of FoxO3a, and translocation

16

of the glucocorticoid receptor to the nuclei in the tibialis anterior muscle in C57BL/6J

17

mice. Oral administration of glabridin prevented dexamethasone-induced expression of

18

MuRF1 and Cbl–b, and phosphorylation of FoxO3a (Figure 6A). Furthermore,

19

glabridin inhibited dexamethasone-induced nuclear translocation of the glucocorticoid

20

receptor (Figure 6B). These in vivo results were consistent with the in vitro ones.

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Discussion

23

In this study, we demonstrated that glabridin, one of the major flavonoids in

24

Glycyrrhiza glabra L., prevented dexamethasone-induced muscle atrophy in C2C12

25

myotubes and C57BL/6J mice. Treatment with glabridin significantly inhibited 17

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dexamethasone-induced protein degradation in C2C12 myotubes (Figure 1A, B).

2

Furthermore, glabridin significantly decreased dexamethasone-induced MuRF1 and

3

Cbl-b expression (Figure 1C). Dexamethasone-induced muscle atrophy functions via

4

two signaling pathways. One is mediated by the activation of the glucocorticoid

5

receptor and another is by the activation of p38/FoxO3a signaling. Glabridin inhibited

6

dexamethasone-induced translocation of the glucocorticoid receptor to the nuclei

7

(Figure 2) through binding to the receptor protein (Figure 3). Although glabridin also

8

inhibited dexamethasone-induced phosphorylation of p38 and FoxO3a (Figure 4), this

9

pathway was not found to be important to inhibit protein degradation as shown by the

10

RNA interference experiment (Figure 5). Finally, we found that oral administration of

11

glabridin inhibited dexamethasone-induced muscle atrophy in mice (Table 1),

12

accompanied by inhibition of dexamethasone-induced expression of MuRF1 and Cbl-b

13

(Figure 6). Glabridin inhibited nuclear translocation of the glucocorticoid receptor and

14

phosphorylation of FoxO3a (Figure 6). These in vivo results were similar to the in vitro

15

ones. Thus, glabridin is an effective compound for inhibition of

16

dexamethasone-induced muscle atrophy.

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The glucocorticoid receptor is activated by binding of steroids

followed by translocation to the nuclei, resulting in induction of muscle atrophy by

19

starvation, renal failure, sepsis, and cancer [33]. Dexamethasone has been used for one

20

of the in vitro atrophy models [34]. It has been reported that mifepristone, one of the

21

steroidal antiprogestogens, is an antagonist for the glucocorticoid receptor [35]. In this

22

study, we found that glabridin inhibited dexamethasone-induced muscle atrophy

23

through inhibiting translocation of the glucocorticoid receptor (Figure 5) through direct

24

binding to the glucocorticoid receptor (Figure 3). These results strongly suggest that

25

glabridin acts as an antagonist of the glucocorticoid receptor. To the best of our

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knowledge, there is no report that food ingredients have antagonistic effects against the

2

glucocorticoid receptor. This is the first finding that glabridin, a food ingredient, acts as

3

an antagonist for glucocorticoid receptor. The structure of glabridin has both prenyl

4

and phenolic hydroxyl moieties. The skeleton of isoflavane does not contribute to

5

binding the glucocorticoid receptor because daidzein did not bind to the glucocorticoid

6

receptor (Figure 3). Thus, the prenyl and phenolic hydroxyl moieties of glabridin might

7

be important for binding to the glucocorticoid receptor. Glabridin also inhibited

8

dexamethasone-induced phosphorylation of p38 and FoxO3a (Figures 4 and 6). It is

9

known that p38 is activated by oxidative stress, biochemical stress and inflammatory

10

responses [36-38]. Glabridin has antioxidant capacity and anti-low density lipoprotein

11

oxidation. [39, 40]. Thus, glabridin might decrease dexamethasone-induced oxidative

12

stress, although this function is not related to muscle atrophy. Dexamethasone is used

13

as an anti-inflammatory agent [41]. Dexamethasone induces certain biochemical stress

14

and oxidative stress resulting in the activation of p38/FoxO3a signaling [42]. Insulin

15

growth factor I stimulates muscle growth by suppressing protein breakdown

16

accompanied by expression of atrophy-related ubiquitin ligases, atrogin-1 and MuRF1

17

[43]. Dexamethasone alone and combination of dexamethasone and glabridin did not

18

affect phosphorylation of mTOR, Akt at Ser473 and Thr308, and FoxO3a at Ser253

19

(Supplementary Figure S1). Furthermore, glabridin inhibited expression of MuRF1 and

20

Cbl-b, but not atrogin-1 (Figure 1C). Glabridin did not completely inhibit

21

dexamethasone-induced muscle wasting and reduction of protein synthesis because

22

glabridin did not inhibit atrogin-1 expression. There is another possibility that the

23

expression level of atrogin-1 is within the normal range, atrogin-1 may not be involved

24

in glabridin-caused suppression of muscle atrophy.

25

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Glabridin prevented dexamethasone-induced muscle atrophy in mice (Table 19

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1). Oral administration of glabridin significantly inhibited the dexamethasone-induced

2

decrease in the tibialis anterior muscle weight and a decreasing tendency in the

3

gastrocnemius muscle weight. The tibialis anterior, gastrocnemius, and quadriceps

4

muscles are white muscles. It was reported that dexamethasone-induced muscle

5

atrophy did not alter the weight of soleus muscle, a red muscle [44]. In this study, the

6

soleus muscle weight was decreased by the injection of dexamethasone, and glabridin

7

did not inhibit the soleus muscle atrophy. 18-β-Glycyrrhetinic acid, which is a

8

constituent of licorice, has been reported as a gap junction/hemichannel blocker [45].

9

Dexamethasone-induced muscular atrophy has been involved in expression of

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1

10

connexin-based hemichannels [46]. Thus, glabridin might inhibit connexin

11

hemichannels.

12

Dietary glabridin was highly absorbed into the intestine without conjugation [47]. Bioavailability differs depending on the chemical structure. Prenylflavonoids are

14

easily incorporated into the body and prenylation enhances cellular uptake of

15

flavonoids in the intestine, resulting in higher bioavailability [48]. In previous study,

16

9.7 mg/kg body weight of glabridin intragastically injected to rats, and the levels of

17

glabridin in the blood and liver 1 h after injection was 28.2 µM and 53.6 nmols/liver,

18

respectively [47]. Although there is no data about glabridin concentration in the

19

muscle, it is expected that a considerable amount of glabridin is delivered to the

20

muscle. In this study, treatment with glabridin at 10 µM to C2C12 myotubes in vitro

21

has high reliability because the mice were injected 40 mg/kg body weight/day

22

glabridin for 2 weeks in vivo. In this study, we did not measure the levels of glabridin

23

in the blood and muscle of mice. Clarification of the bioavailability of glabridin is

24

interesting and important issue in the future.

25

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Antagonists of glucocorticoid receptors at high dose should be harmful and 20

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cause primary adrenal insufficiency and hypocortisolism, also known as Addison’s

2

disease [49]. Glabridin is a major flavonoid in licorice and high contents in the dry

3

weight of roots of up to 0.35% were reported [50]. Licorice has been listed in the USA

4

by the Food and Drug Administration as Generally Recognized as Safe [51]. Thus, it is

5

safe that glabridin, a food ingredient, act as an antagonist for glucocorticoid receptor.

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6

9

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Conclusion

The food ingredient glabridin was found to inhibit dexamethasone-induced protein degradation in C2C12 myotubes and the muscle atrophy in mice. Glabridin

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attenuated dexamethasone-induced expression of MuRF1 and Cbl–b through the

11

inhibition of glucocorticoid receptor activation. Glabridin bound to the glucocorticoid

12

receptor and acted as an antagonist. These findings suggested that glabridin is an

13

effective food ingredient for the prevention of skeletal muscle atrophy.

14 15

Acknowledgments

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This work was supported by the Cross-ministerial Strategic Innovation

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Promotion Program by Cabinet Office, Government of Japan, and JSPS KAKENHI

18

Grant Number 17H00818 (H.A.).

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Absorption and metabolism of 4-hydroxyderricin and xanthoangelol after oral

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administration of Angelica keiskei (Ashitaba) extract in mice, Arch Biochem

21

Biophys. 521 (2012) 71–76.

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49. W. Arlt, B. Allolio, Adrenal insufficiency, Lancet 361 (2003) 1881-1893.

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50. H. Hayashi, S. Hattori, K. Inoue, O. Khodzhimatov, O. Ashurmetov, Field survey

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of Glycyrrhiza plants in Central Asia (3). Chemical characterization of G. glabra

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collected in Uzbekistan, Chem. Pharm. Bull. 51 (2003) 1338–1340. 27

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51. M.C. Peters, J.A. Tallman, T.M. Braun, J.J. Jacobson, Clinical reduction of S.

2

mutans inpre-school children using a novel liquorice root extract lollipop: a pilot

3

study, Eur Arch Paediatr Dent. 11 (2010) 274e8.

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Table 1 Weight of muscles from dexamethasone-induced atrophy in C57BL/6 mice Dex

Dex + Glabridin

Tibialis anterior muscle

0.67±0.04a

0.47±0.02b

0.58±0.01a

Gastrocnemius muscle

1.28±0.06a

1.08±0.02b

1.19±0.05b

Quadriceps muscle

1.61±0.12a

1.29±0.04a

1.50±0.09a

Soleus muscle

0.08±0.01a

0.07±0.00b

0.07±0.00b

Total muscle mass

3.64±0.22a

2.90±0.06c

3.34±0.15a

TE D

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Control

C57BL/6 mice were divided into three groups (n=4): control, dexamethasone alone

EP

(Dex), and dexamethasone and glabridin (Dex+Glabridin). Glabridin (40 mg/kg body weight/day) was orally administrated to the mice for 2 weeks. After 1 week, the mice

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were intraperitoneally injected with dexamethasone (5 mg/kg body weight/day). Data are presented as the mean ± SE (n=4). Different letters indicate significant differences (p < 0.05 by Tukey-Kramer test). 5 6

28

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Figure Legends

2

Figure 1. Glabridin inhibited dexamethasone-induced protein degradation.

3

Differentiated C2C12 myotubes were labeled with 0.04 MBq L-3H-tyrosine for 24 h.

4

The cells were treated with 1 µM dexamethasone for 30 min followed by 0.1, 1 and 10

5

µM of glabridin for 24 h. (A) Radioactivity in the medium was measured and the

6

protein degradation rate was calculated. (B) Radioactivity in the intracellular protein

7

was also measured and the proportion of remaining labeled protein was calculated. (C)

8

C2C12 myotubes were treated with 0.1, 1 and 10 µM of glabridin for 30 min and

9

followed by 1 µM dexamethasone for 24 h. After incubation, the cell lysate was

M AN U

SC

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1

10

prepared and subjected to western blotting to detect the expression level of ubiquitin

11

ligases. Data are presented as the mean ± SE (n=3). Different letters indicate

12

significant differences (p < 0.05 by Tukey-Kramer test).

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13

Figure 2. Glabridin inhibited dexamethasone-induced glucocorticoid receptor

15

translocation to the nuclei. C2C12 myotubes were treated with 10 µM glabridin for

16

30 min followed by 1 µM dexamethasone for 30 min. (A) After the treatment, the

17

nuclear and post nuclear fractions, and cell lysate were prepared and subjected to

18

western blotting for detection of the protein level of the glucocorticoid receptor. (B)

19

After the treatment, immunofluorescence staining was performed. Glucocorticoid

20

receptor and nuclei were stained with Alexa Fluor 488–conjugated secondary anti–

21

rabbit IgG antibody (green) and 4’, 6-diamidino-2-phenylindole (blue), respectively.

22

The numbers of glucocorticoid receptor located in the nuclei were counted, and the

23

percent to the numbers of nuclei was calculated. Data are presented as the mean ± SE

24

(n=3). The intensity of fluorescence on the yellow line in the merged panels was

25

measured. Green, blue, and red lines indicate the glucocorticoid receptor, nucleus, and

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14

29

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1

background, respectively. Different letters indicate significant differences (p < 0.05;

2

Tukey-Kramer test).

3

Figure 3. Glabridin inhibited binding of dexamethasone to the glucocorticoid

5

receptor. (A) The chemical probes were mixed with cell lysate from the C2C12

6

myotubes or recombinant glucocorticoid receptor and incubated for 1 h. (B) Glabridin

7

was mixed with cell lysate from the C2C12 myotubes or recombinant glucocorticoid

8

receptor and incubated for 30 min. The chemical probe was added to the mixture and

9

incubated for another 30 min. After these mixtures were centrifuged at 20,000 × g for 5

10

min at 4°C, the supernatant was collected as a flow-through fraction (F). After the

11

pellet was washed twice, washing solution was referred to as a wash fraction (W). The

12

pellet was collected and used as an eluted fraction (E). Each sample was subjected to

13

western blotting. (C) The tibialis anterior muscle was isolated from C57BL/6J mice.

14

An aliquot of muscle lysate (1.0 mg protein) was incubated with the

15

anti-glucocorticoid receptor antibody overnight at 4°C. Protein A/G PLUS–Agarose

16

(20 µL) was added to the mixture and it was rotated for 1 h at 4°C. After washing, the

17

complex was treated with 10 µM glabridin and rotated for another 30 min at room

18

temperature. The immune–complex was incubated with 2.6 MBq 3H-dexamethasone

19

and rotated for 30 min at room temperature. After washing, the radioactivity of the

20

immune–complex was measured. Data are presented as the mean ± SE (n=4). An

21

asterisk indicates statistical significance from the control treatment (p < 0.05 by

22

student’s t-test)

AC C

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23 24

Figure 4. Glabridin inhibited dexamethasone-induced phosphorylation of p38 and

25

FoxO3a. C2C12 myotubes were treated with 10 µM glabridin for 30 min followed by 30

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1 µM dexamethasone for 30 min. After incubation, the nuclear and post nuclear

2

fractions were prepared and subjected to western blotting for detection of

3

phosphorylation and expression levels of p38 and FoxO3a. Data are presented as the

4

mean ± SE (n=3). Different letters indicate significant differences (p < 0.05 by

5

Tukey-Kramer test).

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1

6

Figure 5. Glabridin inhibited dexamethasone-induced protein degradation

8

through inhibition of glucocorticoid receptor activation. The myotubes were treated

9

with siRNA targeted to the glucocorticoid receptor or p38 using RNAiMAX for 48 h

10

and labeled with 0.04 MBq L-3H-tyrosine for 24 h. The cells were treated with 1 µM

11

dexamethasone for another 30 min followed by 10 µM glabridin for 24 h.

12

Radioactivity in the intracellular protein was measured and the remaining labeled

13

protein proportion was calculated. Data are presented as the mean ± SE (n=3).

14

Different letters indicate significant differences (p < 0.05 by Tukey-Kramer test).

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Figure 6. Glabridin prevented dexamethasone-induced skeletal muscle atrophy in

17

mice. C57BL/6 mice were divided into three groups of 4 each: control, dexamethasone

18

alone, and dexamethasone and glabridin. Glabridin (40 mg/kg body weight/day) was

19

orally administrated to C57Bl/6J mice for 2 weeks. After 1 week, mice were

20

intraperitoneally injected with dexamethasone (5 mg/kg body weight/day). After 2

21

weeks, the nuclear and post nuclear fractions, and tissue lysate were prepared and

22

subjected to western blotting. Data are presented as the mean ± SE (n=4). Different

23

letters indicate significant differences (p < 0.05 by Tukey-Kramer test).

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EP

16

24 25

Supplementary Figure S1. Glabridin and dexamethasone did not affect 31

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phosphorylation of Akt, mTOR, and FoxO3a at Ser253. C2C12 myotubes were

2

treated with 0.1, 1, or 10 µM glabridin for 30 min followed by 1 µM dexamethasone

3

for 30 min. After incubation, the cell lysate was prepared and subjected to western

4

blotting for detection of the phosphorylation and expression levels of Akt, mTOR, and

5

FoxO3a. Data are presented as the mean ± SE (n=3). Different letters indicate

6

significant differences (p < 0.05 by Tukey-Kramer test).

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4 ｂ

40

0

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30 20 10

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a

a

in cells (%)

a

6

50

3H-tyrosine-labeled

∆Protein degradation (%)

a

protein

A

1

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0 Dex (µM)

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C MuRF-1/GAPDH

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3.0 Cbl-b/GAPDH

b

Atrogin-1/GAPDH

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b

a

0.5

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ab

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0 Dex (µM)

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a

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Dex (µM)

-

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Glabridin (µM)

-

-

0.1

1

10

Glabridin (µM)

-

-

0.1

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10

Yoshioka, Y., et al., Figure 1

A Nuclei Post nuclei ACCEPTED MANUSCRIPT

Whole

GR LaminB GAPDH 1

1

-

1

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-

Glabridin (μM)

-

-

10

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c 20 a 0 -

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Glabridin (μM)

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a

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GR/GAPDH (whole)

1.5

10

Dex (μM)

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Dex (μM)

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1.0

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GR/LaminB (in nuclei)

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Dex (μM)

0.5

0

Dex (μM)

-

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1

Glabridin (μM)

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-

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Continued Yoshioka, Y., et al., Figure 2

Continued B Dex DexMANUSCRIPT + Glabridin ACCEPTED 120

b

100 80 60 40

c

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Dex (µM)

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Nuclear translocation (%)

GR

Control

Yoshioka, Y., et al., Figure 2

A

B W E Glabridin + － + MANUSCRIPT － + － ACCEPTED Glabridin probe + － + － + － Glucocorticoid receptor

β-actin

β-actin

Daizein probe Glabridin probe

Input

F

W

Input

Glabridin Glabridin probe

－ + － + － + + － + － + －

GR

W

E

－ + － + － + + + + + + +

(DPM)

1000

2000

*

1000

+

+

0 3H-Dex

-

+

Glabridin

EP

Glabridin

GR recombinant

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*

500 0 3H-Dex

3000

+

+

-

+

AC C

3H-Dex/GR

(DPM)

1500

Tissue lysate

3H-Dex/GR

C

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－ + － + － + + + + + + +

F

E

W

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Glucocorticoid receptor

F

Input

Daidzein probe Glabridin probe

Input

F

Yoshioka, Y., et al., Figure 3

A

p-FoxO3a (Ser7)

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FoxO3a p-FoxO1

-

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1

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1

Glabridin (μM)

-

-

0.1

1

10 1.5

2.5 b ab a a

a

1.0 0.5 0

Dex (μM)

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0 Dex (μM)

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3.0

-

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1

10

b

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c

2.0 1.5

a

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Post nuclei

p-p38 p38

1

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Nuclei

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LaminB GAPDH

Dex (μM)

-

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p-FoxO3a (Ser7)/LaminB

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p-FoxO1/FoxO1

2.0

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p-FoxO3a at Ser7/FoxO3a

Dex (μM)

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FoxO1

4.0

b

3.0 c 2.0

a

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-

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1

Glabridin (μM) 10 Yoshioka, Y., et al., Figure 4

A

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c c

45

cb

ab ab

ab a

ab

ab

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a

35

～ ～ 0

1

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-

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+

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+

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50

Yoshioka, Y., et al., Figure 5

A p-FoXO3a at Ser7 ACCEPTED MANUSCRIPT

Cbl-b

FoXO3a

Atrogin-1

p-FoXO1

GAPDH

FoXO1

RI PT

MuRF-1

-

5

5

Dex (mg/kgBW)

-

5

5

Glabridin (mg/kgBW)

-

-

40

Glabridin (mg/kgBW)

-

-

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a

2 1

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AC C

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b

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b

p-FoxO3a at Ser 253 /FoxO3a

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4

SC

Dex (mg/kgBW)

0

1

0.5

0 -

5

5

-

5

5

-

-

40

-

-

40

Continued Yoshioka, Y., et al., Figure 6

Continued B

ACCEPTED MANUSCRIPT Nuclei Post nuclei GR LaminB

-

5

5

-

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5

Glabridin (mg/kgBW)

-

-

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-

-

40

1.5

3

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GR/GAPDH

a a

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1

M AN U

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a

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GR/LaminB

b

SC

Dex (mg/kgBW)

RI PT

GAPDH

Yoshioka, Y., et al., Figure 6