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Effect of 1-deoxynojirimycin on cholesterol effluxthrough ABCA1-LXRa pathway in 3T3-L1 adipocytes

1756-4646/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.jff.2013.12.016

* Corresponding author at: Department of Food and Nutrition and Institute of Health Sciences, Korea University, Seoul 136-703of Korea. Tel.: +82 2 940 2857.

E-mail address: mjshin@korea.ac.kr (M.-J. Shin).Abbreviations: DNJ, 1-deoxynojirimycin; HDL, HDL-cholesterol; ABCA1, ATP-binding cassette transporter A1; LXRa, liver X

RXR, retinoid X receptor

Hyun Ju Doa,b, Seung-Min Leec, Young Soon Kima, Min-Jeong Shina,b,d,*

aDepartment of Food and Nutrition, Korea University, Seoul 136-703, Republic of KoreabDepartment of Public Health Sciences, Graduate School, Korea University, Seoul 136-703, Republic of KoreacDepartment of Food and Nutrition, College of Human Ecology, Yonsei University, Seoul 120-749, Republic of KoreadKorea University Guro Hospital, Korea University, Seoul 152-703, Republic of Korea

A R T I C L E I N F O A B S T R A C T

Article history:

Received 12 September 2013

Received in revised form

12 December 2013

Accepted 16 December 2013

Available online 10 January 2014

Keywords:

1-Deoxynojirimycin

ABCA1

LXRa

ApoA1

Cholesterol efflux

3T3-L1 adipocytes

We aimed to investigate whether 1-deoxynojirimycin (DNJ) has effects on cholesterol efflux

in adipocytes and modulates ABCA1 expression in high fat fed mice. Fully differentiated

3T3-L1 adipocytes were tested for the cytotoxicity of DNJ and cholesterol efflux into

apoA1/HDL was then assessed. ABCA1 protein levels and nuclear abundance of LXRa were

measured after the treatment of DNJ in differentiated 3T3-L1 adipocytes. The effects of

12 week DNJ supplementation on ABCA1 gene expression in epididymal adipose tissue of

high fat (HF) fed mice were further evaluated. Overnight treatment of DNJ increased choles-

terol efflux mediated with ApoA1 and HDL in 3T3-L1 adipocytes. DNJ increased the protein

expression of ABCA1 gene and enhanced the nuclear presence of LXRa, the transcription

factor for ABCA1 in 3T3-L1 adipocytes. DNJ supplementation for 12 weeks upregulated

the expression of ABCA1 in epididymal adipose tissue in HF fed mice, however, the effect

was not related to the increase in circulating HDL-cholesterol levels. Thus, DNJ appears

to have a positive impact on the reverse cholesterol metabolism by activating the

ABCA1–LXRa pathway and enhancing the cholesterol efflux. DNJ may decrease the risk of

metabolic disorders related to abnormal regulation of reverse cholesterol transport.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Dysregulated function of adipocytes is related to the risk of

developing metabolic disorders including obesity, inflamma-

tion, cardiovascular diseases, hyperlipidemia, atherosclero-

sis, and insulin resistance (Le Lay et al., 2004; van de

Woestijne et al., 2011; Zhang et al., 2010). In addition,

adipose tissue is one of the most important organs in

cholesterol metabolism and contains 20% of the total body

cholesterol (Wu & Zhao, 2009) and cholesterol efflux via adi-

pocyte can influence the homeostasis of cholesterol metabo-

lism (Chung et al., 2011; Yu et al., 2010). During reverse

cholesterol transport, cholesterols are emitted out from

blood plasma through acceptors such as ApoA1 outside of

adipocytes (Christopher & Phoebe, 2001) and the mature

HDL-cholesterol existing in plasma will move to the liver

through blood stream and inside liver cells through accep-

tors existing in the membrane (Silver et al., 2000). Since

, Republic

receptor a;

J O U R N A L O F F U N C T I O N A L F O O D S 7 ( 2 0 1 4 ) 6 9 2 – 6 9 9 693

elevated blood cholesterol is a well-known risk factor for

atherosclerosis and low HDL level is highly related to the

atherosclerotic risk (McGillicuddy et al., 2011), modulation

of HDL is an effective way to treat atherosclerosis, proven

by drugs targeting HDL levels (von Eckardstein et al., 2001).

Recently, ATP-binding cassette transporter A1 (ABCA1) was

demonstrated to play an important regulatory role in

cholesterol efflux in adipocyte (Zhang et al., 2010). ABCA1 is

a membrane protein that directly provides cholesterol or

phospholipid to ApoA1 in ApoA1 mediated active pathway

(Bortnick et al., 2000; Buechler & Bauer, 2012; Howard et al.,

2010; Vedhachalam et al., 2007), of which the expression is

regulated by liver X receptor a (LXRa) (Cui et al., 2011). Adipo-

cytes regulate the ABCA1 expression levels in order to main-

tain cholesterol homeostasis by affecting the process of the

reverse cholesterol transport (Chung et al., 2011). It was also

demonstrated that lack of ABCA1 in adipocytes indirectly in-

creases the catabolism of HDL, and decreases the level of HDL

in the serum (Brinton et al., 1991).

Many drugs have proven to exert their effects through

mediating enhanced reverse cholesterol transport (Tall,

2008; von Eckardstein et al., 2001). 1-Deoxynojirimycin

(DNJ), originally recognized as a-glucosidase inhibitor

(Tsuduki et al., 2009), is often used to treat patients with

type 2 diabetes mellitus (T2DM) (Bollen et al., 1988; Kim

et al., 2010; Kimura et al., 2007; Nakagawa et al., 2007). Its

biological roles appear to be extended further, for instance,

in Gaucher’s diseases (Abian et al., 2011) and hepatitis (Wat-

son et al., 2001). In addition, recent studies have directly

demonstrated that AMP-DNM which is a DNJ inducer, influ-

ences fat formation, inflammation, and insulin resistance in

animal fat organs (van Eijk et al., 2009). In the present study,

we hypothesized that DNJ might influence ABCA1 gene

expression and reverse cholesterol mechanism in adipo-

cytes, which ultimately exerts beneficial effects against ath-

erosclerotic process by increasing HDL-cholesterol in the

blood. To this end, we assessed the roles of DNJ on ABCA1

and LXRa gene expressions and its effects on ApoA1/HDL

mediated cholesterol efflux in 3T3-L1 adipocytes. Further-

more, we examined the effect of 12 week DNJ supplementa-

tion on ABCA1 gene expression in adipose tissue of high fat

fed mice.

2. Materials and methods

2.1. Chemicals

1-Deoxynojirimycin (DNJ), Isobutyl-1-methylxanthine, dexa-

methasone, and insulin were obtained from Sigma–Aldrich

(St. Louis, MO, USA). 3T3-L1 cells were obtained from the

American Type Culture Collection (ATCC, Manassas, VA,

USA). High-glucose Dulbecco’s Modified Eagle’s Medium

(DMEM) was purchased from Hyclone (Thermo Scientific,

Waltham, MA, USA). Fetal bovine serum (FBS), bovine calf ser-

um (BCS) and antibiotics (100,000 Unit/L penicillamine,

100 mg/L streptomycin) were purchased from Gibco (Carls-

bad, CA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltet-

razolium bromide (MTT) was purchased from Amresco

(Solon, OH, USA).

2.2. Cell culture and adipocyte differentiation

Mouse 3T3-L1 cells were grown in high-glucose DMEM sup-

plemented with 10% heat-inactivated BCS containing

100,000 units/L penicillin, and 100 mg/L streptomycin. Prior

to the experiments, 1 · 105 cells were seeded on a 6-well plate

and grown to confluence for 4 d. At day 5 post-confluence, the

cells were subjected to the first differentiation medium

(DMEM, 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine, 1 lM

dexamethasone, 10 lg/mL insulin) starting on day 0, for

68 h. The medium was replaced with the second differentia-

tion medium (DMEM, 10% FBS, 10 lg/mL insulin). Two days la-

ter, the cells were grown in regular medium (DMEM, 10% FBS,

10 lg/mL insulin) for an additional 2 days. The cells were

maintained at 37 �C in a humidified atmosphere of 95% air

and 5% CO2. The growth medium was replenished every two

days.

2.3. MTT assay

DNJ was prepared in a DMSO solution at a concentration of

1 mM. Fully differentiated 3T3-L1 cells were tested with

increasing concentrations of DNJ for 24 h. Cell viability was

analyzed after the addition of 0.1 mg/mL MTT in each well

and an another additional incubation for 1 h at 37 �C. After re-

moval of the medium, cells were lysed with DMSO. The absor-

bance was detected at 570 nm of wavelength using a

microplate reader.

2.4. Immunoblot analysis

The cells were scraped from the plates with lysis buffer

(40 mM HEPES pH 7.5, 120 mM NaCl, 1 mM ethylenediamine-

tetraacetic acid (EDTA), 1% Triton X-100) containing a prote-

ase inhibitor cocktail (Roche, Basel, Switzerland). After

incubation on ice for 30 min, cell lysates were centrifuged at

18,000g for 45 min at 4 �C. Nuclear proteins were extracted

using the NE-PER* Nuclear and Cytoplasmic Extraction Kit

(Thermo Scientific, Waltham, MA, USA). Proteins were quan-

tified using the BCA protein assay (Sigma–Aldrich, St. Louis,

MO, USA). Protein lysates were resolved on 10% sodium-dode-

cyl sulphate polyacrylamide gels (SDS–PAGE) and then trans-

ferred to a polyvinylidenedifluoride (PVDF) membrane. Anti-

ABCA1 (Abcam, Cambridge, MA, USA), anti-LXRa (Abcam,

Cambridge, MA, USA), anti-b-actin (Santa Cruz, Dallas, TX,

USA) antibodies were used to detect ABCA1, LXRa, and b-actin

respectively. Blots were observed by western blot detection kit

(Ab Frontier, Seoul, Korea). Protein bands were quantified

using the Alphaview� software (Alpha Innotech, San Leandro,

CA, USA).

2.5. Cholesterol efflux assay

The 3T3-L1 cells were seeded in 96-well plate. After differen-

tiation, cells were cultured in the serum-free DMEM (0.5% FBS,

1% antibiotics) overnight (16–18 h). Cells were treated with

NBD-cholesterol (10 lM) and the plates sealed with foil to

block light. After 24 h, the media was removed and cells were

incubated in serum-free DMEM (0.5% FBS, 1% antibiotics) for

694 J O U R N A L O F F U N C T I O N A L F O O D S 7 ( 2 0 1 4 ) 6 9 2 – 6 9 9

6 h. Cells were treated with DNJ. After 24 h, cells were washed

twice with 0.2% BSA–PBS and then incubated in 0.2% BSA–PBS

containing 40 lg/ml ApoA1 (Calbiochem, San Diego, CA, USA)

and 100 lg/ml HDL (Calbiochem, San Diego, CA, USA) for 1 h,

according to Zhang et al. (2011). One hundred microlitres of

the supernatant in each well were collected and transferred

to a 96-well black plate. Residual medium in each well was re-

moved by tapping. Cells were lysed with lysis buffer (5 mM

Tris–HCl pH 7.6, 0.1% SDS) and homogenized on shaker for

10 min. 100 lL of the lysate in each well was collected and

transferred to a 96-well black plate. Fluorescence from the

supernatant (A) and the cell lysate (B) were measured at exci-

tation and emission wavelengths of 485 and 535 nm, respec-

tively. Cholesterol efflux was calculated as (A)/[(B) + (A)].

2.6. Animals and study design

Male C57BL/6 mice (5 weeks old; initial weight 16.3–18.3 g)

were purchased from DBL (Eumseong, Korea) and randomly

assigned into three groups after a 1 week adaptation period:

normal control diet (CTL, n = 10), high fat diet (HF, n = 9), and

high fat diet supplemented with 1-deoxynojoromycin (DNJ,

n = 9). DNJ isolated from Bacillus subtilis MORI (Lee et al.,

2013a) was prepared in distilled water and administered oral-

ly on the basis of body weight (10 mg/kg, once daily). The mice

in CTL and HF groups received equivalent volume of vehicle

(distilled water). The CTL was based on the AIN-76 rodent diet

composition. The HF was identical to the CTL, except that

200 g fat/kg (170 g lard plus 30 g corn oil) and 1% cholesterol

were added to it (Table 1). They were housed under standard

(18–24 �C, 50–60% humidity) laboratory conditions, main-

tained on a 12/12 h light/dark schedule (lights on at

8:00 a.m.), with free access to food and water for 12 weeks.

All the experimental procedures were approved by the Com-

mittee on Animal Experimentation and Ethics of Korea Uni-

versity (Protocol #KUIACUC-2013-95, May 30, 2013). At the

end of the experimental period, mice were fasted overnight

(12 h). Mice were anesthetized with zoletil and rompun (40

Table 1 – Composition of control and high fat test diets (g/kg diet).

Ingredient Control diets High fat diets

Corn starch 150 150

Casein 200 200

Sucrose 500 340

Corn oil 50 30

Mineral mixa 35 35

Vitamin mixb 10 10

Cellulose 50 50

DL-methionine 3 3

Choline bitartrate 2 2

Lard 170

Cholesterol 10

BHTc 0.01 0.01

Total 1000 1000

a AIN-76 mineral mix.b AIN-76 vitamin mix.c Dibutylated hydroxytoluene.

and 5 mg/kg body weight, respectively, i.p.). Blood samples

were obtained from the abdominal inferior vena cava to

analyze HDL. Plasma concentrations of total cholesterol,

HDL-cholesterol and LDL-cholesterol were measured using

Olympus AU400 Chemistry Analyzer (Tokyo, Japan). White

adipose tissues (WAT) were extracted and weighted, then rap-

idly frozen with liquid nitrogen and stored in the freezer at

�80 �C.

2.7. RNA extraction and quantitative real time polymerasechain reaction (qRT-PCR)

Total RNA were extracted from WAT using an RNeasy Lipid

Tissue Mini Kit (Qiagen, Hilden, Germany) according to the

manufacturer’s protocol. The cDNA was synthesized from

1 lg of RNA using oligo-dT and Superscript II reverse trans-

criptase (Invitrogen, Carlsbad, CA, USA). Primer sequences

for PCR are the following: ABCA1, 5 0-TGAAGCCTGTCCAG-

GAGTTC-30 (forward) and 5 0-ATGACAAGGAGGATGGAAGC-3 0

(reverse); ABCG1, 5 0-CAAGACCCTTTTTGAAAGGGATCTC-30

(forward) and 5 0-GCCAGAATATTCATGAGTGTGGAC-3 0 (re-

verse); GAPDH, 5 0-AACTTTGGCATTGTGGAAGG-3 0 (forward)

and 5 0-ACACATTGGGGGTAGGAACA-3 0 (reverse). The real-time

PCR (Step One Plus, Applied Biosystems, Foster City, CA, USA)

conditions were: 15 min at 95 �C, followed by 40 cycles of 94 �Cfor 30 s, 56–59 �C for 20 s and 72 �C for 30 s. GAPDH was used

as the control in the comparative CT method.

2.8. Statistical analysis

Statistical analysis was performed using SPSS. The results

were presented as means ± SE. The differences among the

experimental groups were analyzed using a student t-test

(for in vitro experiments) and one-way analysis of variance

(ANOVA) with Duncan’s multiple range (for animal experi-

ments) with p < 0.05 as the criterion of significance.

3. Results

3.1. The influence of DNJ on cell viability in 3T3-L1adipocytes

In order to assess the cell viability rate, 3T3-L1 adipocytes

were treated with DNJ with concentration levels of 0, 0.1,

0.25, 0.5, and 1 lM (Fig. 1). The 3T3-L1 adipocytes had a viabil-

ity of 87% in 0.1 lM concentration level, and viabilities of 94%,

97%, and 96% at concentration levels of 0.25, 0.5, and 1 lM,

respectively.

3.2. ABCA1 gene expression by DNJ in 3T3-L1 adipocytes

ABCA1 is a component of ATP-binding cassette family protein

and a key transporter involved in cholesterol efflux (Bortnick

et al., 2000). We investigated whether DNJ had an effect on the

ABCA1 gene expression in the 3T3-L1 adipocytes by treating

with various concentrations of DNJ level (Fig. 2). The ABCA1

protein expression demonstrated a significant increase at

the 0.1 lM concentration level (p = 0.02) and was the highest

at a dose of 0.25 lM (p = 0.04) and no further significant in-

crease with the 0.5 lM.

0.0

0.5

1.0

1.5

DMSO 0.1μM 0.25μM 0.5μM

Rel

ativ

e le

vels

of A

BC

A1

prot

ein

expr

essi

on

p=0.04

p=0.03

DNJ

β-actin

ABCA1

DMSO 0.1μM 0.25μM 0.5μM

DNJ

Fig. 2 – Effect of DNJ on the expression of ABCA1 gene in

3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were

treated with DNJ for 24 h. Immunoblot analysis was

performed with anti-ABCA1 antibody. Representative blot is

shown in the upper panel. Bar graph represents

quantification of the relative protein levels of ABCA1.

Results were expressed as means and SE of at least five

independent experimental results. P < 0.05 by t-test.

J O U R N A L O F F U N C T I O N A L F O O D S 7 ( 2 0 1 4 ) 6 9 2 – 6 9 9 695

3.3. DNJ influencing the cholesterol efflux in 3T3-L1adipocytes

DNJ treatment significantly increased the ApoA1/HDL-medi-

ated cholesterol efflux in 3T3-L1 adipocytes in all tested con-

centrations (Fig. 3). A 13% increase was noted at the 0.1 lM

concentration level (p = 0.03), and a 16% increase was noted

at the 0.25 lM concentration level (p = 0.01), while a 15% in-

crease was noted at 0.5 lM concentration level (p = 0.03). As

for the HDL mediated cholesterol efflux, DNJ effect was con-

centration-dependent with a 14% increase at 0.25 lM concen-

tration level (p = 0.003) and a 22% increase at the 0.5 lM

concentration level (p = 0.006).

3.4. LXRa gene expression by DNJ in 3T3-L1 adipocytes

Since the LXRa regulates the expression of ABCA1, we exam-

ined its expression at both total protein and nuclear protein

levels. As shown at Fig. 4, The LXRa gene expression in nucle-

ar protein showed an increased tendency at all concentration

levels of 0.1, 0.25, and 0.5 lM without reaching a statistical

significance. There was no difference in LXRa gene expression

in total protein.

3.5. Effects of DNJ supplementation on ABCA1 geneexpression of adipose tissue and circulating HDL-cholesterolin high fat fed mice

We further tested whether 12 week DNJ supplementation on

HF could influence mRNA abundance of ABCA1 and ABCG1

in epididymal adipose tissue of HF fed mice. The results

showed that HF significantly reduced the expression of

ABCA1 in epididymal adipose tissue, which was increased

by DNJ supplementation (Fig. 5A). Similarly, DNJ supplemen-

tation increased the expression of ABCG1 (Fig. 5B) in HF fed

mice, which did not reach a significance. Regarding the ef-

fects of DNJ supplementation on blood lipid profile, the levels

of LDL-cholesterol were significantly reduced by DNJ supple-

mentation on HF (Fig. 5E). On the other hand, DNJ supplemen-

tation did not affect plasma levels of HDL-cholesterol

(Fig. 5D).

0.0

0.2

0.4

0.6

0.8

1.0

1.2

DMSO 0.1μM 0.25μM 0.5μM 1μM

MT

T as

say

DNJ

Fig. 1 – Cell viability effect of DNJ on 3T3-L1 adipocytes was

measured by MTT assay. Average value of three

independent experiments is shown in graph.

4. Discussion

Previously, the role of DNJ in adipose tissue was examined

only in terms of its effects on insulin sensitivity via modulat-

ing adipose tissue metabolism (Aerts et al., 2007; van Eijk

et al., 2009). In the current study, we provide evidence show-

ing the effects of DNJ on promoting the reverse cholesterol

transport in adipocytes. Adipocytes are important in choles-

terol metabolism (Le et al., 2004; Zhang et al., 2010) and adipo-

cytes can modulate cholesterol efflux as a way to regulate the

homeostasis of cholesterol in the body (Yu et al., 2010). As

demonstrated by Yancey et al. (2003), the increase in

cholesterol efflux could lead to positive effects in reducing

the risk of developing atherosclerosis. In the present study,

overnight treatment of DNJ increased cholesterol efflux med-

iated with ApoA1 and HDL in 3T3-L1 adipocytes. To gain an

insight on the molecular mechanism of DNJ effect in choles-

terol efflux in adipocytes, we examined the expression levels

of ABCA1 gene and its related transcription factor, LXRa. Ear-

lier studies have showed a strong relationship between the

expression of ABCA1 and cholesterol efflux (Bortnick et al.,

2000; Buechler & Bauer, 2012; Howard et al., 2010; Vedhacha-

lam et al., 2007). ABCA1, a transmembrane transporter in-

volved in cholesterol efflux, has been well characterized in

macrophages (Howard et al., 2010) and recently in adipocytes

(Verghese et al., 2007; Zhang et al., 2010). The role of ABCA1 in

cholesterol efflux in adipose tissue was also implicated in an

animal study using ABCA1 knockout mice (Chung et al., 2011).

Our data showed that DNJ increased ABCA1 expression in

3T3-L1 differentiated adipocytes with the concentration of

0.0

0.5

1.0

1.5

DMSO 0.1μM 0.25μM 0.5μM

Cho

lest

erol

eff

lux -

Apo

A1

0.0

0.5

1.0

1.5

DMSO 0.1μM 0.25μM 0.5μM

Cho

lest

erol

eff

lux-

HD

LA

B

DNJ

DNJ

p=0.01

p=0.03

p=0.03

p=0.003

p=0.006

Fig. 3 – Effects of DNJ on ApoA1/HDL induced cholesterol

efflux in 3T3-L1 adipocytes. (A) ApoA1 induced cholesterol

efflux (B) HDL induced cholesterol efflux; differentiated 3T3-

L1 adipocytes were incubated in serum free DMEM

containing NBD-cholesterol (10 lM) for 24 h, and were

treated DNJ for 24 h. ApoA1 (40 lg/ml) and HDL (100 lg/ml)

were added and the cells and medium were collected after

1 h. Radioactivity of medium and cell lysates was counted.

Results were expressed as means and SE of at least three

independent experimental results. P < 0.05 by t-test.

Total β-actin

Total LXRα

DMSO 0.1μM 0.25μM 0.5μM

DNJ

Nuclear β-actin

Nuclear LXRα

Fig. 4 – Effect of DNJ on the expression of LXRa gene in 3T3-

L1 adipocytes. Differentiated 3T3-L1 adipocytes were treated

with DNJ for 24 h. (A) Total protein extract (B) Nuclear protein

extract; Immunoblot analysis was performed with anti-

LXRa antibody. Representative blot is shown in the upper

panel. Bar graph represents quantification of the relative

protein levels of LXRa. Results were expressed as means

and SE of at least five independent experimental results.

P < 0.05 by t-test.

696 J O U R N A L O F F U N C T I O N A L F O O D S 7 ( 2 0 1 4 ) 6 9 2 – 6 9 9

0.1 lM or higher. This effect of DNJ could be exerted by affect-

ing the activity and/or levels of transcription factor, LXRa for

ABCA1 expression. LXR functions as a cholesterol sensor sup-

pressing the rise of cholesterol by regulating the expression of

ABCA1 and ABCG5/ABCG8 (Kimura et al., 2007; Zhang et al.,

2010). In the present study, the DNJ-treated adipocyte showed

elevated levels of LXRa in the nucleus upon the treatment of

0.1 lM or higher. However, there was no significant increase

in the amount of total LXRa in the cell. Therefore, DNJ is likely

to partition LXRa into the nucleus without affecting the total

protein levels of LXRa. Similar to our observations, LXRa was

shown to be regulated its subcellular localization according to

the glucose levels (Helleboid-Chapman et al., 2006). This dif-

ferential intracellular partitioning of LXRa could be another

way to regulate its transcriptional activity in the nucleus.

Even if more detailed experiments need to be done, we have

shown that DNJ was able to increase nuclear fraction of LXRa,

which could lead to an increase in ABCA1 expression in DNJ-

treated adipocytes. Previously, another plant-derived compo-

nent, curcumin was shown to have effects on cholesterol ef-

flux. Curcumin effects were mediated by promoting PPARc-

mediated signaling pathway in adipocytes according to the

study by Dong et al. (2011). PPARc is a transcription factor

for LXRa and increases ABCA1 expression by increasing the

expression levels of LXRa (Dong et al., 2011; Wu & Zhao,

2009). However, DNJ did not show observable changes in pro-

tein levels of LXRa. Therefore DNJ-induced cholesterol efflux

may not be elicited by affecting PPARc.

In the present study, we further tested whether DNJ sup-

plementation for 12 weeks could modulate the expressions

of ABCA1 and ABCG1 in adipose tissue of high fat fed mice,

which ultimately influence HDL-cholesterol in blood. Emerg-

ing evidence has shown that cholesterol efflux in adipose

tissue influences plasma HDL, thereby plays an important

role against atherosclerotic process (McGillicuddy et al.,

2011). Indeed, it was recently reported that ABCA1-depen-

dent cholesterol efflux in adipose tissue and nascent HDL

particle formation contribute to systemic HDL biogenesis,

suggesting that adipose tissue ABCA1 expression plays an

important role in adipocyte cholesterol homeostasis (Chung

et al., 2011). Our results demonstrated significant upregula-

tions of ABCA1 expression in epididymal adipose tissue with

12 week DNJ supplementation. Taken together with our re-

sults in vitro, we can speculate that DNJ might promote the

formation of nascent HDL as shown by an increase in the

levels of cholesterol efflux after the addition of apoA1 or

HDL. On the other hand, there was no observable difference

in plasma levels of HDL-cholesterol between HF and DNJ

groups, indicating the elevated ABCA1 gene expression was

not enough to elicit HDL-increasing effects in this setting.

Further studies investigating clinical effect of DNJ on HDL

biogenesis and circulating HDL-cholesterol levels are war-

ranted. Accumulating data have reported food components

which show functional properties in reverse cholesterol

0

30

60

90

CTL HF DNJ

HD

L-c

hole

ster

ol

(mg/

dL)

ab

0.0

3.0

6.0

9.0

12.0

CTL HF DNJ

LD

L -c

hole

ster

ol

(mg/

dL)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

CTL HF DNJ

Rel

ativ

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vels

of A

BC

G1

mR

NA

exp

ress

ion

0.0

0.2

0.4

0.6

0.8

1.0

1.2

CTL HF DNJ

Rel

ativ

e le

vels

of A

BC

A1

mR

NA

exp

ress

ion

b

aab b

NS

b

aaA B

C D

0

30

60

90

120

150

180

CTL HF DNJ

Tot

al c

hole

ster

ol

(mg/

dL)

E

ba

ab

Fig. 5 – Effects of DNJ administration (10 mg/kg, p.o., once daily) on mRNA expressions of ABCA1 (A), ABCG1 (B) in epididymal

adipose tissue and plasma total cholesterol (C), HDL-cholesterol (D) and LDL-cholesterol (E). CTL (n = 10): normal control diet,

HF (n = 9): high fat diet, DNJ (n = 9): high fat diet supplemented with DNJ. The results were expressed as means ± S.E. Tested by

one-way ANOVA with Duncan’s multiple range. Values with the same superscript letter are not significantly different (a–c).

J O U R N A L O F F U N C T I O N A L F O O D S 7 ( 2 0 1 4 ) 6 9 2 – 6 9 9 697

efflux and/or circulating HDL cholesterol, including resvera-

trol (Chang et al., 2012), walnut (Berryman et al., 2013), quer-

cetin (Lee et al., 2013b) and quercetin-rich onion peel

extracts in animals (Lee et al., 2012) and humans (Lee

et al., 2011). Considering the protective effect of reverse cho-

lesterol efflux and HDL biogenesis against atherosclerotic

process, DNJ could be used in the development of functional

food potentially useful in preventing cardiovascular disease

and low HDL-associated metabolic disorders.

There are some limitations in the current study. Since we

have not used an antagonist to block LXRa signaling pathway,

it is not clear whether LXRa-mediated effects are the major

events exerted by DNJ treatment. In addition, other transcrip-

tional factors for ABCA1 have not been tested in the study.

Nonetheless, we provide evidence that the treatment of

DNA in fully differentiated 3T3-L1 adipocytes resulted in a

significant increase in ApoA1/HDL-mediated cholesterol ef-

flux. The DNJ-induced upregulation of ABCA1 expression

may be partly mediated by the increased expression in nucle-

ar LXRa. In conclusion, DNJ showed a positive impact on the

reverse cholesterol metabolism by activating the ABCA1-LXRa

pathway and enhancing the cholesterol efflux. DNJ may

decrease the risk of metabolic disorders related to abnormal

regulation of reverse cholesterol transport.

5. Conflict of interest

None.

Acknowledgments

This research was supported by Basic Science Research

Program through the National research Foundation of Korea

(NRF) funded by the Ministry of Education, Science and

Technology (NRF-2013R1A1A2A10006101).

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