Bisdemethoxycurcumin Inhibits Adipogenesis in 3T3-L1

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Bisdemethoxycurcumin inhibits adipogenesis in 3T3-L1 preadipocytes and suppresses obesity in high-fat diet-fed C57BL/6 mice Ching-Shu Lai, Ying-Yi Chen, Pei-Sheng Lee, Kalyanam Nagabhushanam, Chi-Tang Ho, Wen-Shiung Liou, Roch-Chui Yu, and Min-Hsiung Pan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05577 • Publication Date (Web): 16 Jan 2016 Downloaded from http://pubs.acs.org on January 25, 2016

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Journal of Agricultural and Food Chemistry

Table of contents

Lipid accumulation BDMC Adipogenesis

High fat diet Adipocyte size Adipose tissue weight Body weight gain ACS Paragon Plus Environment 1


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Bisdemethoxycurcumin inhibits adipogenesis in 3T3-L1 preadipocytes and

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suppresses obesity in high-fat diet-fed C57BL/6 mice

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Ching-Shu Lai , , Ying-Yi Chen , , Pei-Sheng Lee£, Nagabhushanam Kalyanam ,

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Chi-Tang Ho , Wen-Shiung Liou , Roch-Chui Yu , Min-Hsiung Pan , , ¥,*

5

£

6

Taiwan

7

§

8

Taiwan

9

&

10

¢

11

Ψ

12

Taiwan

13

14

University, Taichung 40402, Taiwan

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¥

16

Taiwan

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#

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Key words: obesity, adipogenesis, bisdemethoxycurcumin (BDMC), mitotic clonal

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expansion (MCE), high-fat diet

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Correspondence:

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*Please send all correspondence to:

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Dr. Min-Hsiung Pan

23

Institute of Food Science and Technology

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National Taiwan University

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No.1, Section 4, Roosevelt Road, Taipei 10617, Taiwan

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Tel. no. (886)-2-33664133; Fax. no. (886)-2-33661771

£ § ,#

£#

¢

&

Ψ

£

£

Institute of Food Science and Technology, National Taiwan University, Taipei 10617,

Department of Seafood Science, National Kaohsiung Marine University, Kaohsiung,

Sabinsa Corporation, 20 Lake Drive, East Windsor, NJ 08520, USA

Department of Food Science, Rutgers University, New Brunswick, NJ 08901, USA Department of Obstetrics and Gynecology, Kaohsiung Veterans General Hospital,

Department of Medical Research, China Medical University Hospital, China Medical

Department of Health and Nutrition Biotechnology, Asia University, Taichung,

These authors contributed equally to this work.

1

ACS Paragon Plus Environment

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E-mail: [email protected]

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Abstract

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Obesity is caused by excessive accumulation of body fat and is closely related to

30

complex metabolic diseases. Adipogenesis is a key process that is required in

31

adipocyte hypertrophy in the development of obesity. Curcumin (Cur) has been

32

reported to inhibit adipocyte differentiation, but the inhibitory effects of other

33

curcuminoids present in turmeric, such as demethoxycurcumin (DMC) and

34

bisdemethoxycurcumin (BDMC), on adipogenesis have not been investigated. Here,

35

we investigated the effects of curcuminoids on adipogenesis and the molecular

36

mechanisms of adipocyte differentiation. Among three curcuminoids, BDMC was the

37

most eﬀective suppressor of lipid accumulation in adipocytes. BDMC suppressed

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adipogenesis in the early stage primarily through attenuation of mitotic clonal

39

expansion (MCE). In BDMC-treated preadipocytes, cell cycle arrest at the G0/G1

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phase was found after initiation of adipogenesis and was accompanied with

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downregulation of cyclin A, cyclin B, p21 and mitogen-activated protein kinase

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(MAPK) signaling. The protein levels of the adipogenic transcription factors

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peroxisome proliferator-activated receptor (PPAR)γ and CCAAT/enhancer-binding

44

proteins (C/EBP)α were also reduced by BDMC treatment. Furthermore, 0.5% dietary

45

BDMC (w/w) significantly lowered body weight gain and adipose tissue mass in

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high-fat diet (HFD)-fed mice. The results of H&E staining showed that dietary

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BDMC reduced hypertrophy in adipocytes. These results demonstrate for the first

48

time that BDMC suppressed adipogenesis in 3T3-L1 adipocytes and prevented

49

HFD-induced obesity. Our results suggest that BDMC has the potential to prevent

50

obesity.

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Introduction

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The prevalence of obesity has become a major global health challenge1. Obesity is

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defined by the WHO as abnormal or excessive fat accumulation that may produce

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adverse health consequences. Numerous studies indicate that obesity is an important

56

risk factor for type 2 diabetes, cardiovascular disease, fatty liver disease, cancers and

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premature death2. The fundamental cause of obesity is an energy imbalance between

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energy intake and energy expenditure3. Adipose tissue is the main storage site of

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excess energy in the form of triacylglycerols that result in both hypertrophy (increase

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in cell size) and hyperplasia (increase in cell number) of adipocytes4. Therefore,

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inhibition of adipogenesis or adipocyte differentiation could be an effective strategy

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for treating or preventing obesity and related diseases.

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Adipogenesis is the multi-step process by which preadipocytes differentiate into

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mature adipocytes that involves growth arrest of confluent preadipocytes, MCE, and

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terminal differentiation5,6. It has been demonstrated that the MCE, characterized by

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growth-arrested preadipocytes synchronously re-entering the cell cycle and increasing

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cell number7, is an essential process for differentiation and adipogenesis of 3T3-L1

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adipocytes8. Differentiation of preadipocytes is tightly regulated by a cascade of

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cellular signaling and transcription factors such as C/EBPs and PPARγ, which

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modulate the gene expression for lipogenesis and accumulation of lipid droplets9.

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A number of signaling pathways have been identified in adipogenesis including

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Kruppel-like factors (KLFs), Wingless/INT-1 proteins (Wnts), cell cycle proteins, and

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insulin-dependent signaling5. In the early stage of adipocyte differentiation, activation

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of PI3K/AKT and MAPK signaling are critical for cell proliferation, differentiation

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and survival10. Extracellular signal-regulated kinase (ERK) is also found to

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phosphorylate and translocate into the nucleus to initiate MCE in preadipocytes

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during the early stage of differentiation11. In addition, tyrosine phosphorylation of 4



78

IRS-1 activates Akt, initiating MCE in adipocytes by amplification of downstream

79

signaling cascades12. Because MCE is required for preadipocyte differentiation,

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interruption of the signaling involved in MCE, such as the ERK and Akt pathways,

81

may be beneficial in preventing adipogenesis13.

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Turmeric (Curcuma longa L.) is widely consumed as a dietary supplement or as an

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ingredient in South Asian cuisine. Turmeric extract contains three curcuminoids—Cur

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(≈80% relative abundance), DMC (≈15%), and BDMC (≈5%)14, which have different

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methoxy substitutions on the aromatic ring15. Multiple preclinical studies indicate that

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curcumin extract from the rhizomes of the turmeric plant exerts antioxidant,

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anti-inflammatory, and antineoplastic effects as well as other beneficial biological

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activities16. Several studies have demonstrated the anti-obesity effects of Cur

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including the inhibition of adipocyte differentiation and obesity-related diseases17,18.

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Although Cur is considered a promising chemotherapeutic agent, preclinical and

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clinical studies have shown that curcumin has limited therapeutic applications due to

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its instability in physiological conditions. Meanwhile, attention has turned to both

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BDMC and DMC because they are more stable than curcumin in physiological

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medium19, which indicates that they may have therapeutic potential under

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physiological conditions. In this present study, we investigated the inhibitory effects

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of BDMC on differentiation of 3T3-L1 preadipocytes and HFD-induced obese mice

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and explored the potential molecular mechanisms underlying these effects.

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Materials and methods

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Chemicals

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Dulbecco's modified Eagle's medium (DMEM), penicillin–streptomycin, fetal bovine

101

serum (FBS), and fetal calf serum (FCS) were purchased from Gibco BRL (Grand

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Island, NY, USA). Insulin, 3-isobutylmethylxanthine (IBMX), dexamethasone (DEX),

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and propidium iodide (PI) were purchased from Sigma Chemical Co. (St. Louis, MO,

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USA). Rosiglitazone was purchased from Cayman Chemical (Ann Arbor, USA).

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The PI3K, p-PI3K, Akt, p-Akt, JNK, p-JNK, P38, p-P38, ERK1/2, p-ERK1/2

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PPARγ, C/EBPα, CDK Antibody Sampler Kit, and Cyclin Antibody Sampler Kit were

107

purchased from Cell Signaling Technology (Beverly, MA, USA). The p21 antibody

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was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The mouse

109

β-actin monoclonal antibody was purchased from Sigma Chemical Co. Cur was

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purchased from Merck (Kenilworth, NJ, USA), DMC and BDMC were obtained from

111

Sabinsa Corp. (East Windsor, NJ, USA).

112 113

Cell Culture and Adipocyte Differentiation

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3T3-L1 mouse preadipocytes were purchased from the American Type Culture

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Collection (Rockville, MD, USA) and were cultured in Dulbecco’s modified eagle

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medium (DMEM) high glucose supplemented with 10% fetal calf serum (FCS) at 37

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°C under a humidified 5% CO2 atmosphere until confluence. The 3T3-L1

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preadipocytes were differentiated based on the method described in our previously

119

study

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incubated in differentiation medium (DMI) containing DMEM, 10% fetal bovine

121

serum (FBS), 0.5 mM 3-isobutyl-1-methylxanthine, 1 µM dexamethasone, 5 µg/mL

122

insulin, and 2 mM rosiglitazone. After 2 days, the medium was replaced with DMEM

20

. Briefly, full confluent 3T3-L1 preadipocytes (defined as Day 0) were

6



123

containing 10% FBS and 5 µg/mL insulin, and the medium waschanged every 2 days.

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The cells were fully differentiated into mature adipocytes on Day 8.

125 126

Cytotoxicity Assays

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The effect of BDMC on cell viability of 3T3-L1 adipocytes was analyzed by the

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trypan blue assay. Differentiation of 3T3-L1 preadipocytes and BDMC treatment were

129

described above. At the differentiation (Day 2), the cells were harvested, and

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cytotoxicity was determined by trypan blue exclusion and microscopy examination.

131 132

Oil Red O Staining

133

For Oil Red O staining, the cells were washed twice with PBS, fixed with 10%

134

formalin overnight, and then stained with 0.5% filtered Oil Red O solution for 30 min.

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Excess Oil Red O staining solution was removed and the cells were washed twice

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with distilled water and dried. The stained lipid droplets were visualized by light

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microscopy and photographed with a digital camera at 400× magnification. The lipid

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droplets stained with Oil Red O were eluted with 100% isopropanol and quantified by

139

measuring the absorbance at 510 nm by spectrophotometer.

140 141

Cell Cycle Analysis

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The cell cycle analysis was based on the method described in our previous study 20.

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Briefly, postconfluent 3T3-L1 cells were cultured in DMI medium with or without

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BDMC for 18 and 24 h. The cells were then harvested, washed with PBS,

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resuspended in PBS, and fixed in 99% ice-cold ethanol at -20 °C. Fluorescence

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emitted from the PI–DNA complex was quantified after excitation of the fluorescent

147

dye by FACScan cytometry (Becton Dickinson, San Jose, CA, USA). The analysis of

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cell cycle distribution was performed with Modfit 4.0 Software (Becton Dickinson). 7


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Protein Extraction and Western Blotting

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The protein extraction and Western blotting analysis was performed as our previously

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study20. Briefly, differentiated cells were harvested and lysed in ice-cold lysis buffer

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for 30 min, followed by centrifugation at 10000 × g for 30 min at 4 °C. The protein

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concentrations were measured by using the Bio-Rad Protein Assay kit (Bio-Rad

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Laboratories, Munich, Germany). Equal amount of protein for each sample (50 µg)

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was subjected to SDS–polyacrylamide gel electrophoresis and transferred to PVDF

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membranes (Millipore Corp., Bedford, MA, USA). The membranes were blocked

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with blocking solution containing 5% bovine serum albumin (BSA) and then

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incubated with the indicated primary antibodies at 4°C overnight, and subsequently

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with HRP-conjugated secondary antibodies (Cell Signaling, Beverly, MA, USA) for 1

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h at room temperature. The HRP activity was visualized by using the VisGlow™

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Chemiluminescent Substrate, HRP (Visual Protein, Taipei, Taiwan), and the density of

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the protein bands was quantified using ImageJ imaging software (National Institutes

163

of Health).

164 165

Animal Experiments

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Four-week-old male C57BL/6J mice were purchased from the BioLASCO

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Experimental Animal Center (Taiwan Co., Ltd., Taipei, Taiwan). The mice were

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housed in a room maintained at 25 ± 1°C with 50% relative humidity and 12 h of

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light/dark cycle, and were with free access to water and the Purina 5001 diet (LabDiet,

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PMI Nutrition International) for 1 week. All animal experimental protocols used in

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this study were approved by the Institutional Animal Care and Use Committee of the

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National Taiwan University (IACUC, NTU). Mice were randomly distributed into

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five dietary groups (n= 6 per each group): normal diet (ND, 15% energy as fat), HFD

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(45% energy as fat), and HFD supplemented with 0.1% Cur and 0.1 or 0.5% BDMC 8



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(1 or 5 g Cur or BDMC/kg diet), respectively, for 15 weeks. The composition of the

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experimental diet was based on the Purina 5001 diet as described previously20. Food

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consumption and the body weight were recorded daily and weekly, respectively. At

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the end of the experiments, all animals were fasted overnight and sacrificed by CO2

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asphyxiation. Blood, liver, spleen, kidney, and adipose tissues were immediately

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collected, weighed, and photographed.

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Biochemical Analysis

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Blood samples were collected centrifuged at 1,000 ×g for 15 min at 4°C to obtain the

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serum and stored at -80°C until analysis. The glutamate oxaloacetate transaminase

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(GOT),

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concentrations in serum were measured with colorimetric slides (Fujifilm, Kanagawa,

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Japan) by using the biochemistry analyzer (Fujifilm Dri-Chem 3500s; Fujifilm)

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according to the manufacturer’s instructions.

glutamate

pyruvate

transaminase

(GPT)

and

triacylglycerol

(TG)

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Histopathological Examinations

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The perigonadal adipose tissue was dissected and fixed in 10% buffered formalin.

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Fixed tissues were processed for embedding in paraffin and 5 µm sections were

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prepared, stained with hematoxylin and eosin (H&E), and subjected to

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photomicroscopic observation. Adipocyte size was measured at 200× magnification

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according to our previously study 21.

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Statistical Analysis

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Statistical evaluate was performed by running the one-way analysis of variance

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(ANOVA) or one-way Student’s t test and Duncan’s Multiple Range Test. Data are

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presented as the means ± SE for the indicated number of independently performed 9


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experiments. A probability value of P Cur. Moreover, only BDMC significantly reduced lipid

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accumulation at concentrations of 10 µM among the three curcuminoids. This result

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suggested that BDMC has more potent anti-adipogenic effects than Cur and DMC.

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BDMC inhibited DMI-induced lipid accumulation in 3T3-L1 adipocytes

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We next examined the cytotoxic effect of BDMC on 3T3-L1 preadipocytes by

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trypan blue assay. As shown in Fig. 2A, BDMC showed no significant cytotoxicity in

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preadipocytes. Fig. 2B illustrates the results of Oil Red O staining under a microscope.

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BDMC potently reduced lipid accumulation in 3T3-L1 adipocytes in a

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dose-dependent manner by 10–65% compared to the DMI-treated group. These

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results demonstrated that BDMC strongly reduced adipogenesis in 3T3-L1 adipocytes

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without affecting cell viability.

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The inhibitory effects of BDMC mainly occurred in the early stage of adipocyte

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differentiation

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It has been shown that 3T3-L1 adipocyte differentiation is initiated by exposure to

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the DMI and undergoes three distinct stages, including the early stage (Days 0–2), the

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postmitotic intermediate stage (Days 3–4) and the terminal stage (after Day 4) 22. To 11


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clarify the molecular mechanisms underlying BDMC-inhibited adipogenesis, we

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examined the effect of 25 µM BDMC at different stages of cellular differentiation as

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indicated in Fig. 3A. After treatment, the accumulation of intracellular lipid droplets

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in adipocytes was determined by quantitative analysis of Oil Red O-stained cells (Fig.

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3 B). We found that BDMC dramatically inhibited DMI-induced lipid accumulation

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during 0–8 (treatment C), 0–6 (treatment D) and 0–4 (treatment E) days. 3T3-L1

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adipocytes treated with BDMC only in the early stage (treatment F) also significantly

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reduced lipid accumulation. A slight inhibitory effect was found with BDMC

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treatment at the middle stage (treatment G and H) but not in the later stage. Because

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MCE is an important step and required for differentiation and adipogenesis, these

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results indicate that BDMC may suppress adipogenesis by inhibiting MCE in the early

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stage of differentiation.

243 244

BDMC repressed DMI-induced cell cycle progression during the MCE process of

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differentiation

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To further examine the effect of BDMC on MCE during adipogenesis, cell cycle

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distribution of 3T3-L1 preadipocytes treated with DMI with or without BDMC was

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analyzed. The results showed that BDMC-treated cells displayed a delayed or blocked

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cell cycle progression at both 18 and 24 h after induction of differentiation with DMI.

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(Fig. 4A) The population of cells in each stage of the cell cycle was quantified (Fig.

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4B). Compared with the undifferentiated group, a significant portion of DMI-treated

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3T3-L1 preadipocytes re-entered into the S phase and G2/M phase at 18 and 24 h,

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respectively. Meanwhile, the cells treated with both DMI and 25 µM BDMC did not

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undergo cell cycle progression and were arrested in G1 phase at both 18 and 24 h.

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Thus, our results demonstrated the effect of BDMC on the suppression of

12



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adipogenesis in 3T3-L1 adipocytes through interference with MCE in the early stage

257

of differentiation.

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Because the above results indicated that BDMC delayed entry of 3T3-L1 cells into

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S phase induced by DMI, we further examined the effect of BDMC on expression of

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proteins involved in the cell cycle. DMI-treated 3T3-L1 adipocytes showed an

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increase in expression of cyclin A and B at 24 h whereas this was significantly

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repressed by BDMC treatment (Fig. 4C). Levels of p21 were increased by BDMC

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treatment, while CDK2, CDK4, cyclin D and cyclin E were not affected. These results

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suggest that BDMC impaired DMI-mediated MCE through downregulation of cyclin

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A and cyclin B and upregulation of p21, which are essential for G1/S and S/G2 phase

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transition of the cell cycle. These results supported the data obtained by FACS

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analysis indicating G1 arrest was induced by BDMC.

268 269

BDMC inhibited the expression of C/EBPα and PPARγ in 3T3-L1 adipocyte

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differentiation

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The PPARγ and C/EBPα pathways play essential roles in adipocytes differentiation

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through regulation of transcription of various genes responsible for lipid transport and

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accumulation 7. Thus, we next evaluated the effects of BDMC treatment on the

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protein expression of these two critical transcription factors. As shown in Fig. 5A, the

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markedly increased protein levels of C/EBPα and PPARγ were observed in

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differentiated adipocytes compared with preadipocytes on Day 4, but these increases

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were dramatically reduced by BDMC at 25 µM (Fig. 5). The results indicated that

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BDMC inhibited the protein expression of PPARγ and C/EBPα that was upregulated

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during adipocyte differentiation. This effect may be a consequence of the BDMC

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inhibition of MCE, which in turn further suppressed terminal adipocyte

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differentiation. 13


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The effects of BDMC on DMI-induced MAPKs and PI3K-Akt signaling during

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adipogenesis

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To elucidate the molecular mechanisms by which BDMC inhibited DMI-induced

286

adipogenesis, the MAPKs and PI3K/Akt pathways were examined by Western blot.

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Intracellular MAPK signaling pathways play a major role in the regulation of cell

288

proliferation and differentiation8. There are three groups of kinases that belong to the

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MAPK family, ERKs, JNKs and p38 MAPK, and the activation of ERK has been

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shown to be essential for the induction of MCE and adipogenesis11,23. We therefore

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assessed whether BDMC inhibited adipogenesis via blockade of the MAPK pathways.

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3T3-L1 preadipocytes were treated with DMI and BDMC, and the phosphorylation of

293

MAPKs was examined by Western blot. The results showed that BDMC treatment

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decreased the phosphorylation of ERK1/2 and JNK but not p38 (Fig. 6A). The

295

phosphorylation of Akt was also reduced by BDMC treatment compared with the

296

DMI group (Fig. 6B).

297 298

BDMC prevented HFD-induced obesity

299

We further investigated the anti-obesity effects of BDMC using a mouse model.

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Mice were fed a HFD with or without BDMC for 15 weeks. Administration of BDMC

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resulted in a less obese phenotype, which may be associated with decreased fat

302

accumulation (Fig. 7A). Mice fed the HFD showed a significantly increased body

303

weight compared to the ND fed group. Furthermore, dietary supplementation of HFD

304

with 0.5% BDMC significantly reduced both final body weight and body weight gain

305

after 15 weeks of feeding compared with those of the HFD mice (Fig. 7B and Table

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1). However, dietary supplementation of HFD with 0.1% BDMC or 0.1% Cur to mice 14



307

showed no significant effect on body weight gain. There were no significant

308

differences in food intake among the BDMC, Cur and HFD group whereas the food

309

efficiency ratio was attenuated by either level of BDMC treatment (Fig. 7C). Dietary

310

BDMC significantly and dramatically reduced the body fat ratio, including

311

perigonadal, retroperitoneal, and mesenteric fat weight (Fig. 8A, 8C and 8D). The

312

analysis of average adipocyte size in epididymal adipose tissue by H&E stain revealed

313

that adipocytes were enlarged in the HFD-fed mice compared to those of the ND mice.

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However, the increase in adipocyte size was significantly smaller in the BDMC

315

treatment mice (Fig. 8B). Moreover, besides slightly increase liver weigh, the relative

316

organ weights of kidney and spleen showed no significant difference between HFD

317

and HFD supplement with 0.1% Cur, 0.1% BDMC and 0.5% BDMC groups (Table

318

2). The mice fed 0.1% and 0.5% BDMC had significantly reduced serum levels of TG,

319

which were similar to the levels in the ND mice (data not shown), indicating that

320

BDMC counteracts the changes to lipid homeostasis caused by the HFD. These

321

results revealed that BDMC was effective in preventing HFD-induced body weight

322

gain and adiposity.

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Discussion

326

There are multiple lines support that prevention and treatment of obesity strategies

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are classified into four categories including reducing food intake, blocking nutrient

328

absorption, increasing thermogenesis, and modulating energy metabolism or storage

329

24

330

fat content of adipocytes, falls into the category of modulating fat storage. The

331

adipocyte life cycle, including proliferation, differentiation and adipogenesis, has

332

been recognized as a potential target for many plant extracts and bioactive compounds

333

for the prevention and treatment of obesity25. The anti-adipogenic and anti-obesity

334

effect of Cur has been widely investigated but other curcuminoids have not been

335

studied. Here, for the first time, we showed that BDMC significantly inhibited

336

adipogenesis in 3T3-L1 preadipocytes and reduced body weight gain in obese mice

337

fed with a HFD.

. Blocking adipocyte differentiation, including reducing adipocyte numbers and the

338

We demonstrated that BDMC markedly inhibited the cytoplasmic lipid

339

accumulation in 3T3-L1 adipocytes with no obvious cytotoxic effects. Significantly,

340

BDMC demonstrated a more potent inhibitory effect than Cur and DMC in

341

DMI-stimulated adipogenesis. BDMC affected DMI-induced adipogenesis in the

342

early stage of differentiation (Fig. 3). Moreover, BDMC slightly decreased lipid

343

accumulation at the middle stage of differentiation of 3T3-L1 adipocytes but had no

344

significant effect in the later stage, suggesting that the inhibition by BDMC primarily

345

occurred in the early stage. We further demonstrated that BDMC inhibited the MCE

346

process in the early stage of adipocyte differentiation. The mechanism by which

347

BDMC suppressed MCE was observed to delay cell cycle progression with a

348

significantly decreased number of cells in the S phase and G2/M phase after DMI

349

induction as well as downregulated cyclin A and cyclin B in the early stage of

350

adipocyte differentiation (Fig. 4). Collectively, these results indicated that BDMC 16



351

suppressed adipogenesis through impairment of the MCE process during the early

352

stage of adipocyte differentiation.

353

Adipocyte differentiation is tightly controlled by a number of signaling pathways

354

and transcription factors. PPARγ and C/EBPα are the key transcription factors which

355

regulate the numerous transcriptional pathways involved in adipocyte differentiation

356

and adipogenesis6,7. Here, we showed that BDMC significantly reduced lipid

357

accumulation in 3T3-L1 adipocytes and effectively blocked adipocyte differentiation

358

by suppressing induction of adipogenic transcription factors such as PPARγ and

359

C/EBPα. The PI3K/Akt signaling is important in transducing the pro-adipogenic

360

effects of insulin. Previous studies have reported that insulin-mediated PI3K/Akt

361

signaling is required for upregulation of PPARγ and induction of adipogenesis26,27.

362

MAPKs also play a pivotal role in many cellular processes including adipocyte

363

differentiation. Among the members of MAPKs, ERK1/2 phosphorylation is required

364

for the expression of the adipogenic transcription factors PPARγ and C/EBPα11,23.

365

Our results revealed that the phosphorylation of ERK1/2 and Akt was reduced by

366

BDMC treatment in 3T3-L1 adipocytes (Fig. 6), which may be associated with the

367

downregulated PPARα and C/EBPα as well as decreased lipid accumulation.

368

However, this requires further investigation. Moreover, a previous report found that

369

the absence of JNK resulted in decreased adiposity and significantly improved

370

cellular sensitivity to insulin which suggested that JNK is crucial in obesity and

371

insulin resistance as well as a potential target for therapeutics28. Our results also

372

showed that BDMC treatment abolished the phosphorylation of JNK, suggesting that

373

BDMC may improve insulin resistance in adipocytes. Therefore, downregulation of

374

ERK1/2, JNK and Akt signaling may one of the mechanisms of BDMC inhibition of

375

adipogenesis.

17


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376

In addition, we investigated the anti-obesity effects of dietary BDMC in obese mice

377

fed with a HFD. The mice in obesity groups showed higher body and serum

378

triacylglycerol levels compared to normal control group (Fig. 7 and 8). Consistent

379

with our in vitro results, the administration of BDMC at doses of 0.1 and 0.5% to

380

mice fed the HFD revealed significantly reduced body weight, fat pad weights and

381

serum triacylglycerol levels of the mice with no significant change in food intake.

382

Dietary BDMC also reduced the size of adipocytes in epididymal adipose tissue from

383

mice treated with HFD. However, the anti-obesity effect was not observed in

384

HFD-fed mice supplemented with 0.1% Cur. Ejaz et al. showed that supplementation

385

of a HFD with 0.05% Cur reduced body weight and adiposity in C57BL/6J mice 17.

386

The different results may be due to the differences in the composition of the HFD and

387

treatment conditions during the experiment.

388

In summary, in the present study we discovered that BDMC, a polyphenol found in

389

turmeric, effectively inhibited the MCE process in the early stage of adipocyte

390

differentiation. BDMC suppressed adipogenesis in 3T3-L1 adipocytes through

391

modulation of cell cycle proteins and the adipogenic transcription factors C/EBPα and

392

PPARγ, likely through inhibition of PI3K/AKT and MAPKs signaling. Finally, the

393

administration of BDMC to mice with HFD-induced obesity reduced body weight

394

gain, fat pad weight, and adipocyte sizes. Based on these findings, we conclude that

395

BDMC has great potential as a novel agent for the prevention and treatment of obesity.

396

Further research is needed to identify the molecular targets of BDMC.

397

18



398 399

Acknowledgment This study was supported by the National Taiwan University NTU-104R7777;

400

Ministry

of

Science

401

102-2628-B-002-053-MY3

and

Technology

101-2628-B-022-001-MY4,

402 403 404

Conflicts of interest The authors declare no conflicts of interest.

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405

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2002, 420, 333-336.

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494

Figure legends

495

Figure 1. Effect of curcuminoids on lipid accumulation in 3T3-L1 adipocytes. (A)

496

Chemical structures of curcuminoids. (B) Preadipocytes were differentiated in the

497

presence or absence of curcuminoids, and the lipid accumulation was determined by

498

Oil red O staining on Day 8. Cur, DMC and BDMC inhibited lipid accumulation at 25

499

µM. Data are expressed as means ± SE. *p < 0.05, **p < 0.01, ***p < 0.001 vs. DMI

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group. #p < 0.05 vs. Cur 5 µM group. &p < 0.05 vs. Cur 10 µM group. $p < 0.05, $$p

Bisdemethoxycurcumin Inhibits Adipogenesis in 3T3-L1

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