ORIGINAL PAPER

Effect of xanthohumol and isoxanthohumol on 3T3-L1 cellapoptosis and adipogenesis

Jeong-Yeh Yang Æ Mary Anne Della-Fera ÆSrujana Rayalam Æ Clifton A. Baile

Published online: 15 September 2007

� Springer Science+Business Media, LLC 2007

Abstract Xanthohumol (XN), the chalcone from beer

hops has several biological activities. XN has been shown

to induce apoptosis in cancer cells and also has been

reported to be involved in lipid metabolism. Based on these

studies and our previous work with natural compounds, we

hypothesized that XN and its isomeric flavanone, iso-

xanthohumol (IXN), would induce apoptosis in adipocytes

through the mitochondrial pathway and would inhibit

maturation of preadipocytes. Adipocytes were treated with

various concentrations of XN or IXN. In mature adipocytes

both XN and IXN decreased viability, increased apoptosis

and increased ROS production, XN being more effective.

Furthermore, the antioxidants ascorbic acid and 2-mer-

captoethanol prevented XN and IXN-induced ROS

generation and apoptosis. Immunoblotting analysis showed

an increase in the levels of cytoplasmic cytochrome c and

cleaved poly (ADP-ribose) polymerase (PARP) by XN and

IXN. Concomitantly, we observed activation of the effec-

tors caspase-3/7. In maturing preadipocytes both XN and

IXN were effective in reducing lipid content, XN being

more potent. Moreover, the major adipocyte marker pro-

teins such as PPARc, C/EBPa, and aP2 decreased after

treatment with XN during the maturation period and that of

DGAT1 decreased after treatment with XN and IXN.

Taken together, our data indicate that both XN and IXN

inhibit differentiation of preadipocytes, and induce apop-

tosis in mature adipocytes, but XN is more potent.

Keywords Reactive oxygen species � Caspase-3/7 �Mitochondrial membrane potential �Peroxisome proliferator-activated receptor c (PPARc) �CCAAT/enhancer binding protein a (C/EBPa)

Introduction

Obesity is a complex disorder with multiple causes including

both genetic and environmental factors. Therefore, the pre-

vention and treatment of obesity are critical to curtail the

rising incidence of morbidity and mortality. Potential ther-

apeutic agents, especially from natural products, that have

the ability to inhibit adipogenesis or increase cell death by

apoptosis could be important tools in preventing obesity.

One group of compounds with potential anti-obesity activi-

ties are flavonoids, which are either synthetic or natural

constituents of foods or drink (tea) [1, 2]. Xanthohumol (XN)

is the principal flavonoid found in the hop plant, Humulus

lupulus L. (Cannabaceae). XN is largely converted into its

isomeric flavanone, isoxanthohumol (IXN) during the wort

boiling [3]. XN is the most abundant prenylated flavonoid in

hops whereas IXN is the most abundant flavonoid in all types

of beer tested [4]. More recently alternative uses for hop

compounds and their effects on biological processes have

become an area of interest. In particular, XN has been shown

to have cancer-inhibiting properties [5, 6]. Its many biolog-

ical activities include inhibition of PGE2 or NO production

[6], anti-tumor activity in hypoxic tumor cells [7], inhibition

of diacylglycerol acyltransferase-1 (DGAT1) activity and

Drs. Baile and Della-Fera are shareholders and serve on the Board of

Directors of AptoTec, Inc.

J.-Y. Yang � M. A. Della-Fera � S. Rayalam � C. A. Baile (&)

Department of Animal and Dairy Science, 444 Edgar L. Rhodes

Center for Animal and Dairy Science, University of Georgia,

Athens, GA 30602-2771, USA

e-mail: cbaile@uga.edu

C. A. Baile

Department of Foods and Nutrition, University of Georgia,

Athens, GA 30602-2771, USA

123

Apoptosis (2007) 12:1953–1963

DOI 10.1007/s10495-007-0130-4

expression in Raji cells [8], and the induction of apoptosis in

various cell types [9, 10]. In addition, XN has also been

reported to inhibit TG synthesis in hepatocytes [11]. XN has

also been shown to act as a natural ligand for the farnesoid X

receptor (FXR), a member of the nuclear hormone receptor

superfamily. XN has been shown to activate FXR in vitro

and it modulated genes involved in lipid or glucose metab-

olism [12]. Based on these studies and our previous

experience in studying flavonoids, we hypothesized that the

prenylated flavonoids from hops and beer would inhibit

adipogenesis and induce apoptosis in adipocytes, making

them potentially useful as antiobesity agents.

Adipocyte number increases as a result of increased

proliferation and differentiation of preadipocytes [13]. A

decrease in adipose tissue mass involves the loss of lipids

through lipolysis and may also involve the loss of mature

and immature fat cells through apoptosis [14–16]. Adipo-

cyte differentiation is mediated by a series of programmed

changes in gene expression [17]. A cascade of transcription

factors, in particular peroxisome proliferators-activated

receptor (PPAR) families and CCAAT enhancer binding

protein (C/EBP), control the process of adipocyte differ-

entiation. Thereafter the expression of adipocyte genes,

including adipocyte lipid binding protein (aP2) and lipid-

metabolizing enzymes, dramatically increase [18].

Inducers of apoptosis include both intra- and extracel-

lular stimuli, such as DNA damage, disruption of the cell

cycle, detachment of cells from their surrounding tissue,

and loss of trophic signaling [19]. Apoptosis occurs pri-

marily through two well-recognized pathways in cells [20].

Both effector mechanisms of apoptosis are associated with

caspase activation and include the intrinsic, or mitochon-

drial-mediated, pathways and the extrinsic, or death

receptor-mediated, pathways [21]. The intrinsic pathway of

apoptosis relies primarily on the permeabilization of

mitochondrial membranes, with associated release of

apoptotic mitochondrial proteins, leading to activation of

caspase 9 and downstream cleavage of caspase 3, 6, or 7

[22]. The objective of this study was to examine the bio-

chemical mechanism by which XN and IXN induce

apoptosis and inhibit adipogenesis in 3T3-L1 cells. Our

findings indicate that XN was more potent than IXN in

inducing apoptosis in mature adipocytes and in inhibiting

adipogenesis in maturing preadipocytes.

Materials and methods

Cell culture

3T3-L1 mouse embryo fibroblasts were obtained from

American Type Culture Collection (Manassas, VA) and

cultured as described elsewhere [23]. Briefly, cells were

cultured in Dulbecco’s modified Eagle’s medium (DMEM)

(GIBCO, Grand Island, NY) containing 10% bovine calf

serum (BCS) until confluent. Two days after confluency

(D0), the cells were stimulated to differentiate with DMEM

containing 10% fetal bovine serum (FBS), 167 nM insulin,

0.5 lM IBMX, and 1 lM dexamethasone for 2 days (D2).

Cells were then maintained in 10% FBS/DMEM medium

with 167 nM insulin for another 2 days (D4), followed by

culturing with 10% FBS/DMEM medium for an additional

4 days (D8), at which time more than 90% of cells were

mature adipocytes with accumulated fat droplets. All

media contained 100 U/ml of penicillin, 100 lg/ml of

streptomycin, and of 292 lg/ml glutamine (Invitrogen,

Carlsbad, CA). Cells were maintained at 37�C in a

humidified 5% CO2 atmosphere.

Reagents and antibodies

Phosphate-buffered saline (PBS) and DMEM medium were

purchased from GIBCO (BRL Life Technologies, Grand

Island, NY). XN and IXN were purchased from ALEXIS

(San Diego, CA). The viability assay kit (CellTiter 96

Aqueous One Solution Cell Proliferation Assay; containing

3-(4,5-dimethythizol-2-yl)-5-(3-carboxymethoxyphenyl)-

2-(4-sulfophenyl)-2H-tetrazolium assay reagent (MTS)

and Caspase-GloTM 3/7 assay kit were purchased from

Promega (Madison, WI). Ascorbic acid and 2-mercapto-

ethanol were purchased from Sigma (St. Louis, MO, USA).

Antibodies specific for polyclonal peroxisome proliferator-

activated receptor (PPAR)c, CCAAT/enhancer binding

proteins (C/EBPs), aP2, DGAT1, poly(ADP-ribose) poly-

merase (PARP), b-Actin, and cytochrome c were from

Santa Cruz Biotechnology (Santa Cruz, CA).

MTS cell viability assay

Adipocytes were incubated with XN and IXN. Prior to

measuring viability, treatment media were removed and

replaced with 100 ll fresh 10% FBS/DMEM medium and

20 ll MTS solution. Cells were then returned to the incu-

bator for an additional 2 h before 25 ll of 10% SDS was

added to stop the reaction. The absorbance was measured at

490 nm in a plate reader (lQuantTM Bio-Tek Instruments,

Inc. Winooski, VT) to determine the formazan concentra-

tion, which is proportional to the number of live cells.

Apoptosis assays

For the assessment of apoptosis, the ApoStrandTM ELISA

Apoptosis Detection Kit (Biomol, Plymouth Meeting, PA)

1954 Apoptosis (2007) 12:1953–1963

123

was used. This kit detects single stranded DNA, which

occurs in apoptotic cells but not in necrotic cells or in cells

with DNA breaks in the absence of apoptosis [24, 25].

Adipocytes were incubated with XN and IXN for the times

and at the concentrations indicated in the Results and

Figure legends. Thereafter, treatment media was removed

and the cells were fixed for 30 min and assayed according

to the manufacturer’s instructions.

Caspase-3/7 activity assay

Adipocytes were incubated with XN and IXN (times and

concentrations indicated in Results and figure legends).

Thereafter, 100 ll of caspase-Glo 3/7 (Promega, Madison,

WI) reagent was added to each sample and the cells were

incubated for 1 h and assayed according to the manufac-

turer’s instructions.

Measurement of intracellular ROS generation

The determination of ROS generation was based on the

oxidation of the nonfluorescent 2,7-dichlorodihydrofloures-

cein diacetate (DCHF) into a fluorescent dye, 2,7-

dichloroflourescein (DCF) by peroxide. Control cells and cell

treated with XN and IXN (times and concentrations indicated

in Results) were analyzed for changes in fluorescence. Fol-

lowing exposure to treatment, cells were washed twice with

PBS and then incubated for 30 min at 37�C in the dark with

the oxidation sensitive probe, DCHF (Molecular Probes,

Eugene, OR) at 2.5 lM. Production of ROS was measured

by the change in fluorescence at an excitation wavelength of

495 nm and an emission wavelength of 525 nm.

Measurement of mitochondrial membrane potential

Changes in the mitochondrial trans-membrane potential

during apoptosis were measured using 3,30-dihexyloxa-

carbocyanine (DiOC6; Molecular Probes, Eugene, OR),

which is a cationic dye. Adipocytes exposed to XN and

IXN for up to 90 min were incubated with 100 nM DiOC6

for 30 min at 37�C. The cells were then washed twice in

PBS and detached from the plates by trypsinzation. Fluo-

rescence was measured with excitation wavelength of

488 nm and emission wavelength of 530 nm.

Quantification of lipid content and oil red O staining

Lipid content was measured using a commercially avail-

able kit (AdipoRed Assay Reagent; Cambrex Bio Science

Walkersville, Inc.). In brief, XN and IXN along with 0.01%

DMSO control were added with the induction medium for

days 0–6 of adipogenesis. Medium was changed every

2 days. On day 6, intracellular lipid content was measured

by AdipoRed Assay. Cells were washed with PBS (pH 7.4)

and 200 ll of PBS was added to the wells. About 5 ll of

AdipoRed reagent was added to each well. After 10 min,

the plates were placed in the fluorometer and fluorescence

was measured with excitation wavelength of 485 nm and

emission wavelength of 572 nm. To visualize lipid content,

treated cells were stained with oil red O and hematoxylin as

described by Suryawan and Hu [26]. After mounting with

glycerol gelatin, three images for each dish were captured

using ImagePro software (MediaCybernetics, Silver

Spring, MD).

Western blot analysis

Whole cell extracts were prepared by washing the cells

with PBS and suspending in a lysis buffer (20 mM Tris,

pH 7.5, 150 mM sodium chloride, 1 mM EDTA, 1 mM

EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate,

1 mM b-glycerophosphate, 1 mM sodium vanadate

(Na3VO4), 1 lg/ml aprotinin, 1 lg/ml leupeptin, and

100 ug/ml phenylmethylsulfonyl fluoride). After 30 min

of rocking at 4�C, the mixtures were centrifuged

(10,000g) for 10 min, and the supernatants were collected

as whole-cell extracts. To isolate the cytosolic fraction,

cells were washed with ice-cold PBS and resuspended in

isotonic homogenizing buffer (250 mM sucrose, 10 mM

potassium chloride, 1.5 mM magnesium chloride, 1 mM

EGTA, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM

phenylmethylsulfonylfluoride, 1 lg/ml aprotinin, 1 lg/ml

leupeptin, 10 mM HEPES-KOH, pH 7.4). After 30 min

incubation on ice, cells were homogenized with a glass

Dounce homogenizer (30 strokes) and centrifuged at 700g

for 10 min. The supernatant was collected as the cytosolic

fraction. The protein concentration was determined by the

method of Bradford [27] with bovine serum albumin as

the standard. Western blot analysis was performed using

the commercial NUPAGE system (Novex/Invitrogen,

Carlsbad, CA), where a lithium dodecyl sulfate (LDS)

sample buffer (Tris/glycerol buffer, pH 8.5) was mixed

with fresh dithiothreitol and added to samples. Samples

were then heated to 70�C for 10 min, separated by 12%

acrylamide gels and analyzed by immunoblotting as pre-

viously described [28]. Immunoblots were developed

using ECL kit (Piscataway, NJ, USA). All experiments

were repeated at least two times. Representative Western

blots are shown along with the graphs of the quantitative

data.

Apoptosis (2007) 12:1953–1963 1955

123

Quantitative analysis of Western blot data

Measurement of signal intensity on PVDF membranes after

Western blotting with various antibodies was performed

using a FluorChemTMdensitomer with AlphaEaseFCTM

image processing and analysis software (Alpha Innotech

Corporation). For statistical analysis, all data were

expressed as integrated density values (IDV). For PPARc,

C/EBPa, aP2, DGAT1, and PARP, IDVs were calculated

as the density values of the specific protein bands/b-actin

density values and expressed as percentage of the control.

For cytochrome c, IDVs are expressed as percentage of

0 h. All figures showing quantitative analysis include data

from at least three independent experiments.

Statistical analysis

Data were converted to percent control by dividing raw

values of each replicate by the mean control values (sep-

arate means were calculated for controls from each time

period, when applicable). The ratios were then multiplied

by 100. One- or two-way analysis of variance (GLM pro-

cedure, Statistica, version 6.1; StatSoft, Inc.) was used to

determine significance of treatment effects and interac-

tions. Fisher’s post-hoc least significant difference test

was used to determine significance of differences

among means. Statistically significant differences are

defined at the 95% confidence interval. Data shown are

means ± SEM.

Results

XN and IXN reduced cell viability and induced

apoptosis

3T3-L1 mature adipocytes were treated with XN or IXN at

various doses for 24 or 48 h. After treatment, the number

of live cells was determined by the MTS assay. As shown

in Fig. 1, both compounds caused a reduction in cell via-

bility in a dose- and time-dependent manner. At the 75 lM

Fig. 1 Effect of XN and IXN on viability. Mature 3T3-L1 adipocytes

were incubated with XN (A) or IXN (B) at various concentrations (0,

25, 50, 75, 100 lM) for 24 or 48 h. Cell viability was determined by

the MTS colorimetric assay. Assays were performed on eight

replicates for each treatment. abcd: Within a time period means that

are not denoted with a common letter are different, P \ 0.05

Fig. 2 Effect of XN and IXN on apoptosis. Mature 3T3-L1

adipocytes were incubated with XN (A) or IXN (B) at various

concentrations (0, 25, 50, 75, 100 lM) for 24 or 48 h. Cell apoptosis

was evaluated by ssDNA ELISA. Assays were performed on eight

replicates for each treatment and repeated twice. abcd: Within a time

period means that are not denoted with a common letter are different,

P \ 0.05

1956 Apoptosis (2007) 12:1953–1963

123

concentration XN (51.0 ± 2.1% decrease, P \ 0.05) was

more effective than IXN (22.1 ± 1.6% decrease, P \ 0.05)

at 24 h. Half-maximal inhibitory concentrations (IC50) for

XN were calculated using the results obtained from via-

bility data. The IC50 values for XN decreased from

75 ± 5 lM after 24 h to 53 ± 3.5 lM after 48 h. However,

the IC50 values for IXN were not calculated since maximal

inhibition of cell viability was not reached with the con-

centrations tested.

We next investigated whether the reduction in cell

number by both isomers involved apoptosis, using the

ssDNA ELISA assay as an indicator of cellular apoptosis.

As shown in Fig. 2, exposure of adipocytes to XN or IXN

resulted in a dose- and time-dependent induction of apop-

tosis. At 75 lM, XN increased apoptosis by 115.6 ± 9.7%

(P \ 0.05) and 229.5 ± 14.4% (P \ 0.05) after 24 and

48 h, respectively. IXN also increased apoptosis by

64.1 ± 9.1% (P \ 0.05) and 69.2 ± 14.1% (P \ 0.05) after

24 and 48 h, respectively. Half maximal effective con-

centration (EC50) for XN decreased from 73 ± 12.5 lM

after 24 h to 56.5 ± 5 lM after 48 h. The EC50 value for

IXN after 48 h was 80 ± 12 lM. Thus, XN was more

effective than IXN in inducing apoptosis. These results

indicate that the decrease in viability was due, at least in

part, to induction of apoptosis by both isomers. The 75 lM

concentration of XN and IXN was selected for subsequent

experiments.

XN and IXN triggered apoptosis by oxidative stress

Several reports suggest an involvement of ROS in the signal

transduction pathway leading to apoptosis [29]. To determine

the involvement of ROS in XN and IXN-induced apoptosis,

ROS levels were measured in adipocytes treated with the two

compounds. As shown in Fig. 3, the intracellular hydrogen

peroxide levels increased dramatically after cells were trea-

ted with XN. In contrast, IXN induced a slow and smaller

increase in intracellular hydrogen peroxide. After 20 min

incubation of mature adipocytes with XN (Fig. 3A), ROS

was 1509.0 ± 125.1% greater than control while the increase

after treatment with IXN (Fig. 3B) was only 36.9 ± 7.3%.

Furthermore, when adipocytes were pretreated with ascorbic

acid (AA) and 2-mercaptoethanol (2-ME) for 3 h before

incubation with XN or IXN, ROS production was effectively

reduced (Fig. 3C). We next investigated whether the

enhanced apoptosis by both compounds involved changes in

ROS production. AA or 2-ME pretreatment blocked the

Fig. 3 Effect of XN and IXN on intracellular hydrogen peroxide

production. 3T3-L1 adipocytes were incubated with 75 lM XN (A) or

IXN (B) and the time-course on ROS production was determined by

the change in fluorescence of the oxidized probe as indicated in

Materials and Methods. (C) Reduction of XN and IXN-induced ROS

generation in cells pretreated with ascorbic acid (AA) or 2-mercap-

toethanol (2-ME). Peroxide levels measured as described in Materials

and Methods are shown for untreated cells (control); cells exposed to

75 lM XN or IXN for 20 min; cells preloaded with 1 mM AA or 2-

ME for 3 h, and cells preloaded with 1 mM AA or 2-ME in the

absence or presence of 75 lM XN or IXN for 20 min. (D) Reduction

of XN or IXN-induced apoptosis in cells pretreated with AA or 2-ME.

3T3-L1 adipocytes pretreated with 1 mM AA or 2-ME for 3 h prior to

exposure to 75 lM XN or IXN for 24 h. Cellular apoptosis was

evaluated by ssDNA ELISA. Assays were performed in eight

replicates for each treatment and ROS production experiments were

repeated twice. abc: Means that are not denoted with a common letter

are different, P \ 0.05

Apoptosis (2007) 12:1953–1963 1957

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increase in apoptosis induced by both compounds (Fig. 3D).

This indicates that ROS generation is a critical component in

apoptosis induced by both XN and IXN.

XN and IXN decreased mitochondrial membrane

potential and increased cytochrome c release

To evaluate the changes in mitochondrial membrane

potential (Dwm) during the apoptotic process, a strong

cationic dye, DiOC6(3), was used. 3T3-L1 mature adipo-

cytes were treated with 75 lM concentration of XN or IXN

at the indicated times. Treatment with IXN for 90 min had

no effect on the Dwm but XN caused dissipation of the Dwm

in a time-dependent manner (Fig. 4A), as indicated by

changes in DiOC6(3) up-take. As the dissipation of Dwm is

associated with the release of cytochrome c from mito-

chondria, Western blot analyses for cytochrome c were

performed. Treatment of cells with either XN or IXN

increased cytoplasmic cytochrome c levels in a time-

dependent manner (Fig. 4B). XN (335.1 ± 45.5% increase,

P \ 0.05) was more effective than IXN (136.4 ± 21.3%

increase, P \ 0.05) at 48 h.

XN and IXN induced activation of caspase-3/7

To determine whether the observed cytochrome c release

was accompanied by activation of caspase 3/7, we mea-

sured caspase 3/7 activity in mature 3T3-L1 adipocytes.

Caspase 3/7 activity increased in a time-dependent manner

during treatment with either XN or IXN. After 12 h incu-

bation of mature adipocytes with 100 lM XN (Fig. 5A),

caspase 3/7 was 75.0 ± 1.8% greater than control

(P \ 0.05), while the increase after treatment with IXN

(Fig. 5B) was 38.9 ± 4.2% greater than control (P \ 0.05).

XN and IXN induced PARP cleavage

Activation of caspase-3 leads to the cleavage of a number

of proteins, one of which is PARP. We further confirmed

the activation of caspase-3 by observing the cleavage of

PARP. Treatment of 3T3-L1 mature adipocytes with

75 lM concentration of XN or IXN caused dose-and time-

dependent proteolytic cleavages of PARP (116 kDa), with

disappearance of the full-length protein and accumulation

of 85 kDa fragments (Fig. 6).

XN and IXN inhibited lipid accumulation

To determine the effect of XN and IXN on lipid accumu-

lation 2-day post confluent preadipocytes were treated with

XN or IXN for 6 days as described in Materials and

Methods. Results from the AdipoRed assays showed that

both flavanones reduced lipid content in a dose dependent

manner (Fig. 7A and B). At 25 lM, XN (97.9 ± 0.1%

reduction, P \ 0.05) was more potent than IXN

(17.9 ± 3.1% reduction, P \ 0.05). Oil red O staining

Fig. 4 Effect of XN and IXN on mitochondrial membrane potential

and cytoplasmic cytochrome c levels. (A) Time-course of the effect of

XN (A) or IXN (B) on MMP. 3T3-L1 adipocytes were incubated with

75 lM XN or IXN for 10, 30, or 90 min. Cells were treated with

DiOC6(3) and fluorescence intensity was measured as indicated in

Materials and Methods. Assays were performed in eight replicates for

each treatment. abcd: Within a time period means that are not denoted

with a common letter are different, P \ 0.05. (B) 3T3-L1 adipocytes

were incubated with 75 lM XN or IXN for 6, 12, 24, or 48 h. Equal

amounts of protein from cytosolic fraction were analyzed by Western

blotting using specific antibodies. Actin was used as an equal loading

control. Densitometric quantitation of the autoradiograms for cyto-

chrome c was performed. Integrated density values were calculated

and expressed as % 0 h. All experiments were repeated in triplicate.

abc: Means that are not denoted with a common letter are different,

P \ 0.05

1958 Apoptosis (2007) 12:1953–1963

123

indicated that 25 lM XN treatment for days 0–6 com-

pletely inhibited 3T3-L1 lipid accumulation (Fig. 7C) and

cells retained the fibroblastic characteristics. Cell viability

assays were performed in parallel with AdipoRed assays to

determine if the decrease in lipid accumulation was due to

decrease in cell numbers. The data indicated that neither

XN or IXN at the tested concentrations had a significant

effect on cell viability (data not shown).

XN and IXN decreased the expression of PPARc and

C/EBPa as well as aP2 and DGAT1

To determine whether the decrease in lipid accumulation

with XN or IXN was related to adipocyte marker protein

expression, 3T3-L1 cells were treated with XN or IXN

from 0–6 days of differentiation. Quantitative analysis

revealed that IXN treatment did not significantly alter the

expression levels of PPARc, C/EBPa, and aP2 but XN

decreased the expression levels (Fig. 8). Interestingly, the

expression of DGAT1 was significantly decreased by both

XN (70.9 ± 3.0% decrease, P \ 0.05) and IXN

(23.6 ± 8.4% decrease, P \ 0.05).

Discussion

This study elucidates the biological effect of XN and its

isomer, IXN, compounds present in extracts from hops

(Humulus lupulus), on the adipocyte life cycle in 3T3-L1

adipocytes. Our study showed that treatment of 3T3-L1

Fig. 5 Effect of XN and IXN on caspase-3/7 activation. 3T3-L1

adipocytes were treated with XN (A) or IXN (B) at various

concentrations (0, 25, 50, 75, 100 lM) for 3, 6, 12, or 24 h.

Caspase-3 activity was determined by the Caspase-Glo 3/7 lumines-

cent assay. Assays were performed on eight replicates for each

treatment. abc: Within a time period means that are not denoted with a

common letter are different, P \ 0.05

Fig. 6 Effect of XN and IXN on PARP cleavage. 3T3-L1 adipocytes

were treated with 75 lM XN or IXN for 6, 12, or 24 h. Cell lysates

were analyzed by Western blotting as described in Materials and

Methods, and PARP protein bands were detected using a specific

antibody, with b-Actin used as an equal loading control (A).

Densitometric quantitation of the autoradiograms for PARP was

performed for XN (B) and IXN (C) treatments, respectively.

Integrated density values were calculated and expressed as % control.

All experiments were repeated three times. abc: Within analytes,

means that are not denoted with a common letter are different,

P \ 0.05

Apoptosis (2007) 12:1953–1963 1959

123

mature adipocytes with XN or IXN induces apoptosis,

inhibits adipogenesis in a dose-dependent manner, with XN

being the more effective compound. Chemically, XN is a

prenylated chalcone (20,4,40-trihydroxy-30-prenyl-60-meth-

oxychalcone) with an open C-ring and IXN (5-O-methyl-8-

prenylnaringenin), an isomer of XN, lacks the open C-ring.

The biological effects of chalcones depends on their

chemical structure [30]. We speculate that the structural

differences may be one reason for the difference in the

effects of XN and IXN. However, further studies are nee-

ded to demonstrate the reason for the difference in the

potency between these compounds.

Our finding that XN induced apoptosis in adipocytes is

consistent with previous reports that XN and its isomer can

induce apoptosis and inhibit cell proliferation in other cell

types, including prostate epithelial cells, B-chronic lym-

phocytic leukemia, and human cancer cell lines [10, 30,

31]. We examined the nature of XN-triggered apoptotic

signaling in mature adipocytes, focusing on ROS mediated

responses. Oxidative stress has been demonstrated to act as

a stimulator of various cell responses, including apoptosis

[32, 33]. There is considerable evidence that ROS can

induce apoptosis via several pathways, including a mito-

chondria-dependent pathway in various cells and tissues

[34]. The mitochondrion is also a pivotal organelle for the

induction of apoptosis via the intrinsic pathway. Increased

mitochondrial permeability and dissipation of the electro-

chemical gradient or membrane potential (MMP) via

opening of the mitochondrial permeability transition pore

(MTP) triggers cell death by releasing apoptogenic factors

from within the mitochondria, with subsequent cytochrome

c release, apoptosome formation, and ultimately apoptosis

induction [35, 36]. Similarly, numerous reports have

demonstrated that ROS generation is an important con-

troller of subsequent apoptotic biochemical changes,

including MMP changes and caspase-3 activation [37, 38].

Thus, we assume that the generation of ROS may act on the

mitochondria, causing MMP loss and subsequently leading

to apoptosis. We found that XN and IXN increased ROS

production (Fig. 3A and B) and XN was more potent than

IXN. Furthermore, AA or 2-ME pretreatment blocked both

the increase in ROS generation and apoptosis induced by

both compounds, confirming that apoptosis induced by

both compounds is at least partly mediated by ROS. In

addition, our data show that XN decreased MMP and

increased release of cytochrome c in a time dependent

manner. Release of cytochrome c activates apoptotic pro-

tease activating factor 1 (Apaf-1), allowing it to assemble

the multiprotein caspase-activating complex apoptosome

and to bind to and activate procaspase-9 and the down-

stream effector caspase cascade [39]. Furthermore,

activation of caspase-3 leads to the cleavage of a number of

proteins, including poly (ADP-ribose) polymerase (PARP).

The cleavage of PARP is another hallmark of apoptosis

[40]. PARP is a nuclear enzyme that facilitates DNA repair

in response to DNA damage [41]. In this regard, Pan et al.

Fig. 7 Effect of XN and IXN on triacylglycerol accumulation. The

differentiation of 2-day post-confluent 3T3-L1 preadipocytes was

induced by standard adipogenic medium to initiate adipogenesis as

described in Materials and Methods in the absence or presence of XN

(A) or IXN (B) at various concentrations (0, 3.125, 6.25, 12.5, 25 lM)

during the days 0–6. On day 6 lipid content was measured by

AdipoRed assay. All assays were performed on eight replicates for

each treatment. abcde: Means that are not denoted with a common

letter are different, P \ 0.05. (C) On day 6, cellular triglyceride was

stained with oil red O dye and cells were photographed at 200·magnification. The experiment was performed on three replicates for

each treatment. Representative images are shown

1960 Apoptosis (2007) 12:1953–1963

123

showed that XN induced apoptosis by activation of cas-

pase-3, PARP cleavage, and Bcl-2 family protein

expression in human colon cancer cells [9]. Consistent with

the above results, our results indicate that XN and IXN

increased caspase3/7 activity (Fig. 5) and caused time-

dependent proteolytic cleavages of PARP (116 kDa), with

the accumulation of 85 kDa fragments.

The adipocyte differentiation program is regulated by

the sequential expression of transcriptional activators,

mainly PPAR families [42]. During adipocyte differentia-

tion, transcriptional factors such as PPARc and C/EBPs are

involved in the sequential expression of adipocyte-specific

proteins such as glucose transporter (GLUT) 4 and aP2 [18,

43]. Our study showed that treatment of 3T3-L1 cells with

XN or IXN during differentiation suppressed lipid

accumulation in maturing preadipocytes in a dose-depen-

dent manner and that XN was the more effective compound

in inhibiting adipogenesis. The cell volume of adipocytes is

largely dependent on the accumulation of triglyceride

(TG). DGAT catalyzes the final step in the glycerol phos-

phate pathway, considered the major pathway for TG

synthesis [44]. Tabata et al. reported that XN inhibited the

activity of DGAT [8]. XN also inhibited TG and apolipo-

protein B secretion [11]. Similarly, in our study, the

expression of DGAT was significantly decreased by XN

and IXN.

Farnesoid X receptor expression was shown to be pos-

itively correlated with lipid accumulation [45] and XN was

shown to act on FXR and regulate downstream gene

expression in a manner similar to selective bile acid

receptor modulators (SBARM) like guggulsterone and

PUFAs [12]. In fact, we have recently shown that cis-

guggulsterone also increases apoptosis of mature adipo-

cytes and decreases adipogenesis of maturing

preadipocytes [46]. Regulation of adipogenesis by XN

might therefore be at least partly mediated by modulating

FXR target genes like PPARc and C/EBPa similar to other

SBARMs. Moreover, in our study, the adipocyte-specific

proteins PPARc, C/EBPa, and aP2 were down regulated

after treatment with XN whereas IXN treatment did not

significantly alter the expression levels of these proteins.

These results suggested that the decreased adipogenesis

caused by XN was accompanied by a strong inhibition of

the adipocyte specific transcription factors.

Conclusion

This report describes a novel discovery that the treatment

of 3T3-L1 adipocytes with XN or its isomer, IXN, leads

to an enhancement of apoptosis and suppression of adi-

pogenesis. Both XN and IXN caused an increase in

intracellular ROS level leading to decreased MMP and

activation of caspase-3 and 7, subsequently leading to

apoptotic biochemical changes, including PARP cleavage

and cytochrome c release, XN being more potent. Fur-

thermore, AA or 2-ME pretreatment significantly blocked

both the increase in ROS generation and apoptosis

induced by both XN and IXN. Both XN and IXN

decreased adipogenesis during the differentiation period,

XN being more potent. Moreover, the major adipocyte

marker proteins such as PPARc, C/EBPa, aP2, and DGAT

decreased after treatment with XN. Although results from

in vitro experiments cannot be directly extrapolated to

clinical effects, these studies may help in elucidating the

various molecular pathways in adipocytes that are tar-

geted by XN and IXN.

Fig. 8 Effect of XN and IXN on expression of adipocyte marker

proteins. The differentiation of 2-day post-confluent 3T3-L1 preadi-

pocytes was induced by standard adipogenic medium to initiate

adipogenesis as described in Materials and Methods in the absence or

presence of 25 lM XN or IXN during the days 0–6. On day 6, cells

were lysed, separated on 10% NUPAGE and subjected to immuno-

blotting to examine the expression levels of PPARc, C/EBPa, aP2 and

DGAT1. Actin was used as an internal reference for sample loading

control. Densitometric quantitation of the autoradiograms for PPARc,

C/EBPa, aP2 and DGAT1 was performed. Integrated density values

were calculated and expressed as % control. All experiments were

repeated in triplicate. abc: Means that are not denoted with a common

letter are different, P \ 0.05

Apoptosis (2007) 12:1953–1963 1961

123

Acknowledgments This work was supported in part by grants from

AptoTec, Inc., and the Georgia Research Alliance and by the Georgia

Research Alliance Eminent Scholar endowment held by CAB.

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