Effects of Capsaicin on Induction of Apoptosis and Inhibition
of Adipogenesis in 3T3-L1 Cells
CHIN-LIN HSU AND GOW-CHIN YEN*
Department of Food Science and Biotechnology, National Chung Hsing University,
250 Kuokuang Road, Taichung 40227, Taiwan
Currently, at the beginning of the 21st century, obesity has become the leading metabolic disease in
the world. It is a serious health problem in industrialized countries. Previous research has suggested
that decreased preadipocyte differentiation and proliferation and decreased lipogenesis are mech-
anisms to reduce obesity. In the present study, the effects of capsaicin on the induction of apoptosis
and inhibition of lipid accumulation in 3T3-L1 preadipocytes and adipocytes were investigated. The
results demonstrated that capsaicin decreased cell population growth of 3T3-L1 preadipocytes,
assessed with the MTT assay. Flow cytometric analysis of 3T3-L1 preadipocytes exposed to capsaicin
showed that apoptotic cells increased in a time- and dose-dependent manner. Treatment with capsaicin
decreased the number of normal cells and increased the number of early apoptotic and late apoptotic
cells in a dose-dependent manner. The treatment of cells with capsaicin caused the loss of
mitochondria membrane potential (∆Ψm). The induction of apoptosis in 3T3-L1 preadipocytes by
capsaicin was mediated through the activation of caspase-3, Bax, and Bak, and then through the
cleavage of PARP and the down-regulation of Bcl-2. Moreover, capsaicin significantly decreased the
amount of intracellular triglycerides and glycerol-3-phosphate dehydrogenase (GPDH) activity in 3T3-
L1 adipocytes. Capsaicin also inhibited the expression of PPARγ, C/EBPR, and leptin, but induced
up-regulation of adiponectin at the protein level. These results demonstrate that capsaicin efficiently
induces apoptosis and inhibits adipogenesis in 3T3-L1 preadipocytes and adipocytes.
KEYWORDS: Capsaicin; adipogenesis; 3T3-L1 cells; apoptosis; protein expression
Obesity is an important topic in the realm of public health
and preventive medicine, because it is considered to be a risk
factor associated with the genesis or development of various
diseases, including coronary heart disease, hypertension, type
2 diabetes mellitus, cancer, respiratory complications, and
osteoarthritis (1). Currently, at the beginning of the 21st century,
obesity has become the leading metabolic disease in the world
(2). Recent reports have proposed mechanisms to reduce obesity,
including decreased energy/food intake and increased energy
expenditure, decreased preadipocyte differentiation and prolif-
eration, decreased lipogenesis, and increased lipolysis and fat
oxidation (3). The preadipocytes play a key role by differentiat-
ing into mature adipocytes and increasing fat mass. Obesity is
characterized at the cell biological level by an increase in the
number and size of adipocytes differentiated from fibroblastic
preadipocytes in adipose tissue (4). Hausman et al. (5) indicated
that adipogenesis is a process wherein the preadipocytes
differentiate into adipocytes. MacDougald and Mandrup (6) also
indicated the major differentiation programmed is coordinated
by several positive and negative adipogenic molecules, including
a variety of growth factors, cytokines, and hormones.
Capsaicin (8-methyl-N-vanillyl-trans-6-nonenamide) is a
major pungent ingredient in red pepper that is widely used as
a spice (7). The pepper is a potent analgesic, anti-inflammatory
(8), and causes desensitization to different chemical irritants
upon long-term treatment. Capsaicin has been reported to
decrease energy intake (9), decrease the adipose tissue weight,
and decrease the serum triacylglycerol content by enhancing
energy metabolism (10). Capsaicin inhibits the growth of various
immortalized and malignant cells (11) and induces apoptosis
in transformed cells (12). The chemical structure of capsaicin
is shown in Figure 1. Cell apoptosis is important for destruction
of undesired cells during development and homeostasis of
multicellular organisms and is characterized by distinct mor-
phological changes such as plasma membrane blebbing, cell
shrinkage, depolarization of mitochondria, chromatin condensa-
tion, and DNA fragmentation (13). There are two main pathways
leading to apoptosis. The first of these depends on the
participation of mitochondria, and the second is involved in the
interaction of a death receptor with its ligand. Many proteins
are known to be involved in the process of programmed cell
death. Caspase are a family of cysteine proteases that are
activated during the execution phase of the cell apoptotic process
* Author to whom correspondence should be addressed. Phone: 886-
4-22879755. Fax: 886-4-22854378. E-mail: firstname.lastname@example.org.
J. Agric. Food Chem. 2007, 55, 1730−1736
10.1021/jf062912b CCC: $37.00 © 2007 American Chemical Society
Published on Web 02/13/2007
(14). Pro- and antiapoptotic members of the Bcl-2 family
regulate the mitochondria pathway (15). Our previous study
revealed that some phenolic acids (including gallic acid,
o-coumaric acid, m-coumaric acid, and chlorogenic acid) caused
an improved inhibition of cell population growth and induction
of apoptosis in 3T3-L1 preadipocytes (16). However, the
literature regarding the effect of capsaicin on induction of
apoptosis and inhibition of adipogenesis in 3T3-L1 preadipo-
cytes and adipocytes is unclear. In the present study, we
investigated the effects of capsaicin on the induction of
preadipocytic apoptosis and inhibition of adipocytic triglyceride
content. The murine 3T3-L1 cell line was used in this study
due to its widespread use as a cell model for adipose cell biology
research over the course of several decades (17).
MATERIALS AND METHODS
Materials. Capsaicin, MTT dye [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide], propidium iodide (PI), ribonuclease
(RNase), oil red O, 3-isobutyl-1-methylxanthine (IBMX), dexametha-
sone (DEX), insulin (INS), and anti-rabbit or anti-mouse secondary
horseradish peroxidase antibodies were purchased from the Sigma
Chemical Co. (St. Louis, MO). Anti-chicken secondary horseradish
peroxidase antibody was purchased from the Bethyl Laboratories, Inc.
(Montgomery, TX). Dimethyl sulfoxide (DMSO) was purchased from
Merck Co. (Darmstadt, Germany). Dulbecco’s modified Eagle’s
medium (DMEM), bovine calf serum, and antibiotic mixture (penicil-
lin-streptomycin) were purchased from the Gibco BRL Co. (Grand
Island, NY). Anti-capsase-3, anti-PARP [poly(ADP-ribose) poly-
merase], and anti-?-actin antibodies were obtained from Cell Signaling
Technology (Beverly, MA). Anti-Bcl-2, anti-Bax, and anti-Bak antibod-
ies were obtained from Pharmingen (San Diego, CA). Anti-PPARγ
(peroxisome proliferator-activated receptor-gamma) antibody was
obtained from Upstate (Lake Placid, NY). Anti-C/EBPR (CCAAT
enhancer binding protein alpha) antibody was obtained from Santa Cruz
Biotechnology (Santa Cruz, CA). Anti-adiponectin antibody was
obtained from BioVision (Mountain View, CA). Anti-leptin antibody
was obtained from Chemicon (Temecula, CA). Molecular mass markers
for proteins were obtained from Pharmacia Biotech (Saclay, France).
Polyvinyldifluoride (PVDF) membranes for Western blotting were
obtained from Millipore (Bedford, MA). All other chemicals were
Cell Culture. The mouse embryo 3T3-L1 cells (BCRC 60159) were
obtained from the Bioresource Collection and Research Center (BCRC,
Food Industry Research and Development Institute, Hsinchu, Taiwan,
ROC). 3T3-L1 preadipocytes were incubated in culture medium
included DMEM, 10% calf serum, 1.5 g/L sodium bicarbonate, and
100 U/mL penicillin-streptomycin. Adipocyte differentiation was
induced by the adipogenic agents (0.5 mM IBMX, 1 µM DEX, and 1
µM INS) that were added to culture medium. Afterward, the medium
was changed to normal culture medium and was freshly replaced every
48 h. The cells were harvested 8 days after the initiation of differentia-
tion. The cell culture condition was 37 °C in a humidified 5% CO2
MTT Assay. The MTT assay was performed according to the
method of Mosmann (18). 3T3-L1 preadipocytes were plated into 96-
well microtiter plates at a density of 1 × 104cells/well. After 24 h, the
culture medium was replaced by 200 µL serial dilutions (0-250 µM)
of capsaicin and the cells were incubated for 24, 48, and 72 h. The
final concentration of the solvent was less than 0.1% in the cell culture
medium. Culture solutions were then removed and replaced by 90 µL
of culture medium. Ten microliters of a sterile, filtered MTT solution
(5 mg/mL) in phosphate-buffered saline (PBS, pH ) 7.4) was added
to each well to reach a final concentration of 0.5 mg MTT/mL. After
5 h, the unreacted dye was removed, and then the insoluble formazan
crystals were dissolved in DMSO (200 µL/well) and measured
spectrophotometrically in a FLUOstar galaxy spectrophotometer (BMG
Labtechnologies Ltd., Germany) at 570 nm. The cell population growth
percentage (%) was expressed as the percentage of cell growth
compared to the control, and it was calculated by A570nm[capsaicin]/
A570nm[control] × 100. The IC50 was calculated as the capsaicin
concentration under which 50% inhibition of cell population growth
occurred compared to that of untreated controls.
LDH Leakage Assay. The lactate dehydrogenase (LDH) leakage
activity assay was performed using a commercial kit (Sigma Chemical
Co., St. Louis, MO). 3T3-L1 preadipocytes were incubated with 0-250
µM capsaicin for 24-72 h and then analyzed for LDH leakage into
the culture media. The total LDH activity was determined after the
cells were thoroughly disrupted by sonication. The percentage of LDH
leakage was then calculated to determine the membrane integrity. The
amount of LDH leakage was expressed as a percentage of total activity
(activity in the medium)/(activity in the medium + activity of the
cells) × 100.
Cell Apoptosis Analysis by PI Staining. Cell apoptosis was assayed
by the PI (propidium iodide) staining method (19). The 3T3-L1
preadipocytes were stimulated with 0-250 µM capsaicin for 24, 48,
and 72 h, respectively. Briefly, cells were harvested by trypsin-EDTA
(TE) solution (0.05% trypsin and 0.02% EDTA in PBS), washed with
PBS twice, and fixed in 80% ethanol at 4 °C for 30 min, followed by
incubation with 100 µg/mL RNase for 30 min at 37 °C. The cells were
then stained with 40 mg/mL PI for 15 min at room temperature and
subjected to flow cytometric analysis of DNA content using a FACScan
flow cytometer (Becton-Dickinson Immunocytometery Systems USA,
San Jose, CA). Approximately 1 × 104counts were made for each
sample. The percentage of distribution of cell apoptosis was calculated
by CELL Quest software.
Annexin V-FITC/PI Double-Staining Analysis by Flow Cytom-
etry. Annexin V-FITC/PI double staining of the cells was determined
using the Annexin V-FITC kit (ANNEX100F, SEROTEC, U.K.). To
detect early apoptosis, late apoptosis, and necrosis induced by capsaicin,
3T3-L1 preadipocytes (1 × 106cells/dish) were added to a 6 cm dish
and treated for 72 h at 37 °C in 3 mL of culture medium containing
testing agents at final concentrations of 0, 50, 100, and 250 µM. 3T3-
L1 preadipocytes (1 × 105) were then stained for 10 min at room
temperature with FITC-conjugated Annexin V-FITC and PI in a Ca2+-
enriched binding buffer (Annexin V-FITC kit) and analyzed by a
FACScan flow cytometer. Annexin V-FITC and PI emissions were
detected in the FL 1 and FL 2 channels of a FACScan flow cytometer,
using emission filters of 525 and 575 nm, respectively. The Annexin
V-FITC-/PI- population was regarded as normal healthy cells, while
the Annexin V-FITC+/PI- cells were taken as a measure of early
apoptosis, Annexin V-FITC+/PI+ as late apoptosis, and Annexin
V-FITC-/PI+ as necrosis. Approximately 1 × 104counts were made
for each sample. The percentage of distribution of normal, early
apoptotic, late apoptotic, and necrotic cells was calculated using CELL
Mitochondria Membrane Potential (∆Ψm) Assay. Mitochondria
membrane potential was determined by using the MitoPTTM 100 test
kit (Immunochemistry Technologies, Bloomington, MN). 3T3-L1
preadipocytes were seeded in 12-well plates. After 24 h, the cells were
treated with 0-250 µM capsaicin for 6, 12, and 24 h, respectively.
Routine passage consisted of rinsing cells in 12-well plates once with
PBS, followed by harvesting with 0.1 mL of TE solution, addition of
1 mL of culture medium, and thorough dispersion. Aliquots of the
resulting cell suspensions were placed in eppendorf tubes containing 1
mL of the culture medium at a concentration of 1 × 106cells per
eppendorf. After being centrifuged (1000 rpm, 5 min), cells were
incubated with 10 µg/mL JC-1 at 37 °C for 15 min in a humidified 5%
CO2incubator. Cells were collected and washed with 1× assay buffer
Figure 1. Chemical structure of capsaicin.
Inhibitory Effect of Capsaicin on 3T3-L1 CellsJ. Agric. Food Chem., Vol. 55, No. 5, 2007
(MitoPTTM 100 test kit). The cells were resuspended in the same
solution and analyzed by a FLUOstar Galaxy fluorescence plate reader
with an excitation wavelength of 485 nm and an emission wavelength
of 590 nm for red fluorescence. Apoptotic cells generate a lower level
of red fluorescence, and changes in the mitochondria membrane
potential (∆Ψm) can be most accurately assessed by comparing the
red fluorescence of untreated cells to the red fluorescence of those
treated with capsaicin.
Measurement of Caspase-3 Activity. After treatment with capsaicin,
3T3-L1 preadipocytes were collected, washed with PBS, and lysed in
lysis buffer (1% Triton X-100, 0.32 M sucrose, 5 mM EDTA, 10 mM
Tris-HCl, pH 8, 2 mM dithiothione, 10 µg/mL pepstatin A, 2 mM
phenylmethanesulfonyl fluoride, and 10 µg/mL leupeptin) for 20 min
at 4 °C followed by centrifugation (10 000g) for 30 min. Caspase-3
activity was assayed in 50 µL reaction mixtures with a fluorogenic
reporter substrate peptide specific for caspase-3. The substrate peptide
(200 µM) was incubated at 37 °C with cytosolic extracts (50 µg of
total protein) in reaction buffer (100 mM HEPES, 10% sucrose, 10
mM dithiothreitol, 0.1% 3-[(3-chloamidopropyldimethylammonio)-1-
propanesulfonate]. Fluorescence was measured after 2 h (excitation
wavelength, 400 nm; emission wavelength, 505 nm) with a FLUOstar
Galaxy fluorescence plate reader (BMG LabTechologies, GmbH,
Measurement of Triglyceride Content. The 3T3-L1 adipocytes
were harvested 8 days after the initiation of differentiation. Cells were
incubated with 0-250 µM capsaicin for 72 h at 37 °C in a humidified
5% CO2incubator. Cells were collected and lysed in lysis buffer (1%
Triton X-100 in PBS). The total triglyceride content in the cells was
determined using a commercial triglyceride assay kit (DiaSys Diagnostic
Systems GmbH, Holzheim, Germany). The protein concentration was
determined by using a Bio-Rad DC protein assay kit (Bio-Rad
Laboratories, Hercules, CA).
Oil Red O Staining. The oil red O working solution was prepared
as described by the method of Ramirez-Zacarias et al. (20). 3T3-L1
adipocytes were harvested 8 days after the initiation of differentiation.
Cells were incubated with 0-250 µM capsaicin for 72 h at 37 °C in a
humidified 5% CO2incubator. Cells were washed twice with PBS and
then fixed with 10% neutral formalin for at least 20 min at room
temperature. After the 10% neutral formalin was removed, 100%
propylene glycol was added to each well for 3 min. Cells were
decolorized with 60% propylene glycol before staining for 1 h with
the oil red O working solution and then washed exhaustively with water.
The staining dye of cells was extracted with isopropyl alcohol (1 mL/
well) and measured spectrophotometrically in a FLUOstar Galaxy
spectrophotometer (BMG Labtechnologies Ltd., Germany) at 510 nm.
The oil red O-stained material (OROSM) was expressed on a per cell
basis using the cell number determined from similar plates. The relative
oil red O-stained material (OROSM, %) relative to control wells
containing cell culture medium without capsaicin was calculated by
A510nm[capsaicin]/A510nm[control] × 100.
Glycerol-3-Phosphate Dehydrogenase Activity. The 3T3-L1 adi-
pocytes were harvested 8 days after the initiation of differentiation.
Cells were incubated with 0-250 µM capsaicin for 72 h at 37 °C in a
humidified 5% CO2incubator. Cells were carefully washed twice with
ice-cold PBS on 3T3-L1 adipocytes and lysed in 25 mM Tris/1 mM
EDTA, pH 7.5 for measurement of glycerol-3-phosphate dehydrogenase
(GPDH) specific activity. GPDH activity was determined according
to the procedure of Wise and Green (21). Protein concentration was
determined by Bio-Rad DC protein assay kit (Bio-Rad Laboratories,
Hercules, CA). Enzyme activity was expressed as units of activity/mg
Western Blot Analysis. The 3T3-L1 preadipocytes were incubated
with 0-250 µM capsaicin for 12 and 24 h at 37 °C in a humidified
5% CO2incubator. 3T3-L1 adipocytes were incubated with 0-100 µM
capsaicin for 12 and 24 h at 37 °C in a humidified 5% CO2incubator.
Cells were collected and lysed in ice-cold lysis buffer (20 mM Tris-
HCl (pH 7.4), 2 mM EDTA, 500 µM sodium orthovanadate, 1% Triton
X-100, 0.1% SDS, 10 mM NaF, 10 µg/mL leupeptin, and 1 mM PMSF).
The protein concentration of the extracts was estimated with a Bio-
Rad DC protein assay (Bio-Rad Laboratories, Hercules, CA) using
bovine serum albumin as the standard. The caspase-3, PARP, Bak, Bax,
Bcl-2, and ?-actin proteins were assessed in preadipocytes. The PPARγ,
C/EBPR, adiponectin, leptin, and ?-actin proteins were assessed in
adipocytes. The total proteins (50-60 µg) were separated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
using a 12% polyacrylamide gel. The proteins in the gel were then
transferred to a PVDF membrane. The membrane was blocked with
5% skim milk in PBST (0.05% v/v Tween-20 in PBS, pH 7.2) for 1 h.
Membranes were incubated with primary antibody (1:5000) at 4 °C
overnight and then with secondary antibody (1:5000) for 1 h.
Membranes were washed three times in PBST for 10 min between each
step. The signal was detected by using the Amersham ECL system
(Amersham-Pharmacia Biotech, Arlington Heights, IL). The relative
expression of proteins was quantified densitometrically using the
software LabWorks 4.5 and calculated according to the reference bands
Statistical Analysis. Each experiment was performed in triplicate.
The results were expressed as the mean ( SD. Statistical analysis was
performed using SAS software. Analysis of variance was performed
using ANOVA procedures. Significant differences (p < 0.05) between
the means were determined by Duncan′s multiple range tests.
Inhibition of Population Growth in 3T3-L1 Preadipocytes.
To assess whether capsaicin inhibited the population growth of
3T3-L1 preadipocytes, cells were treated with 0-250 µM
capsaicin and the cell population growth was determined by
using an MTT assay. As shown in Figure 2A, capsaicin
decreased the cell population growth in a time- and dose-
dependent manner with an IC50value of 45 µM. LDH activity
in the medium and within cells was also measured in order to
evaluate the influence of cell injuries on 3T3-L1 preadipocytes.
The cytotoxic effect of capsaicin on 3T3-L1 preadipocytes is
Figure 2. Effect of capsaicin on the inhibition of cell population growth
(A) and cytotoxicity (B) in 3T3-L1 preadipocytes. Cells were treated with
0−250 µM capsaicin for 24, 48, and 72 h. Reported values are the
mean ± SD (n ) 3); /p < 0.05 compared with the control.
J. Agric. Food Chem., Vol. 55, No. 5, 2007Hsu and Yen
shown in Figure 2B. Capsaicin caused significant LDH leakage
(p < 0.05) as compared with the control.
Capsaicin-Induced Apoptosis in 3T3-L1 Preadipocytes. To
quantify the degree of apoptosis, the amount of sub-G1DNA
was analyzed by flow cytometry. As shown in Figure 3A, flow
cytometric analysis of capsaicin-mediated cell apoptosis of 3T3-
L1 preadipocytes indicated that the increase of apoptotic cells
occurred in a time- and dose-dependent manner. To quantify
the modes of cell death (apoptosis or necrosis) induced by
capsaicin, 3T3-L1 preadipocytes were treated with capsaicin for
72 h, stained with Annexin V-FITC and PI, and analyzed by
flow cytometry. As shown in Figure 3B, flow cytometric
analysis demonstrated that treatment of cells with capsaicin
decreased the number of normal cells (Annexin V-FITC-/PI-)
in a dose-dependent manner. The apoptotic cells including early
apoptotic (Annexin V-FITC+/PI-) and late apoptotic cells
(Annexin V-FITC+/PI+) were increased in a dose-dependent
manner. When the treatment concentrations were increased, the
percentage of normal cells decreased from 99.8% (control) to
73.1% (250 µM). The percentage of apoptotic cells (including
early apoptotic and late apoptotic) increased from 0.1% (control)
to 26.7% (250 µM).
Collapse of Mitochondria Membrane Potential (∆Ψm).
Figure 4 shows the effect of capsaicin on mitochondria
membrane potential (∆Ψm) in 3T3-L1 preadipocytes. A
significant decrease in fluorescence intensity was observed when
3T3-L1 preadipocytes were treated with capsaicin for 6-24 h.
The results demonstrated that the early damage was due to the
change in mitochondria membrane potential, which may further
activate the intrinsic pathway of apoptosis.
Modulation of Apoptosis-Related Activity and Proteins
by Capsaicin. Caspases are essential for the execution of cell
death by various apoptotic stimuli (22). To monitor the
enzymatic activity of caspase-3 during apoptosis induced by
capsaicin, we used the specific fluorogenic peptide substrate
(Ac-DEVD-MCA) for the detection of caspase-3 activity. As
shown in Figure 5A, activation of caspase-3 was significantly
(p < 0.05) increased in capsaicin-treated cells at 12 and 24 h.
There was an approximately 3.3-fold increase of caspase-3
activity resulting from treatment with 250 µM capsaicin for
The effects of capsaicin on the protein expression of caspase-
3, PARP, Bak, Bax, and Bcl-2 in 3T3-L1 preadipocytes are
shown in Figure 5B. Caspase-3 is a member of the caspase
family that has been shown to play an essential role in apoptosis
induced by a variety of stimuli (23). Capsaicin (250 µM)
significantly (p < 0.05) stimulated caspase-3 expression in a
time- and dose-dependent manner and with a maximal increase
of 2.82-fold after 24 h. However, activation of caspase-3 leads
to the cleavage of a number of proteins, one of which is PARP.
This cleavage leads to its inactivation, thus preventing the futile
DNA repair cycle. Treating cells with 250 µM capsaicin
significantly (p < 0.05) induced PARP cleavage with a maximal
cleavage of 0.37-fold occurring after 24 h. The imbalance of
expression of anti- and proapoptotic protein after the stimulus
is one of the major mechanisms underlying the ultimate fate of
cells in the apoptotic process. It is well-known that the proteins
of the Bcl-2 family play a pivotal role in cells undergoing
apoptosis by interfering with the caspases (24). Capsaicin
resulted in a significant (p < 0.05) increase in Bak and Bax
expression from 1.00 (control) to 2.20 and 3.50 (100 µM, 24
h), respectively. Treating cells with capsaicin significantly
(p < 0.05) decreased Bcl-2 expression from 1.00 (control) to
0.25 (100 µM, 24 h).
Inhibition of Intracellular Triglyceride and GPDH activity
in 3T3-L1 Adipocytes. Figure 6 shows the effect of capsaicin
Figure 3. Flow cytometric analysis of capsaicin-mediated cell apoptosis
in 3T3-L1 preadipocytes. (A) PI stained. Cells were treated with 0−250
µM capsaicin for 24, 48, and 72 h. (B) Annexin V-FITC/PI double stained.
Cells were treated with 0−250 µM capsaicin for 72 h. The percentage of
apoptotic/necrotic cells was calculated by CELL Quest software (mean ±
SD, n ) 3); /p < 0.05 compared with the control.
Figure 4. Effect of capsaicin on mitochondria membrane potential (∆Ψm)
in 3T3-L1 preadipocytes. Cells were treated with 0−250 µM capsaicin for
6, 12, and 24 h. Reported values are the mean ± SD (n ) 3); /p < 0.05
compared with the control.
Inhibitory Effect of Capsaicin on 3T3-L1 Cells J. Agric. Food Chem., Vol. 55, No. 5, 2007
on the inhibition of intracellular triglyceride content and GPDH
activity in 3T3-L1 adipocytes. The results demonstrated that
the inhibition of intracellular triglyceride in 3T3-L1 adipocytes
occurred in a dose-dependent manner when cells were exposed
to capsaicin at the concentrations of 0-250 µM (Figure 6A).
The OROSM showed that cell number in 3T3-L1 adipocytes
was not influenced by the treatment with capsaicin. However,
the addition of capsaicin to 3T3-L1 adipocytes resulted in a
marked decrease of GPDH activity in a dose-dependent manner
Modulation of Adipocyte Differentiation-Related Protein
by Capsaicin. PPARγ is known as a key station protein that is
expressed early in the adipocyte differentiation of 3T3-L1 cells
and prior to C/EBPR (25). The effects of capsaicin on the protein
expression of PPARγ, C/EBPR, adiponectin, and leptin in 3T3-
L1 adipocytes are shown in Figure 7. The protein expression
of PPARγ, C/EBPR, and leptin was decreased in a time- and
dose-dependent manner in 3T3-L1 adipocytes treated with
capsaicin. Treatment of 3T3-L1 adipocytes with capsaicin
induced a significant (p < 0.05) up-regulation of adiponectin
expression in a time- and dose-dependent manners. It was
maximally up-regulated to 1.82-fold after cells were treated with
100 µM capsaicin for 24 h.
Obesity is a chronic, stigmatized, and costly disease that is
rarely curable and is increasing in prevalence throughout most
of the world (26). The 3T3-L1 preadipocyte line has been well
characterized in its ability to undergo complete differentiation
into mature adipocytes (27). Adipose tissue consists of adipo-
cytes, which store triacylglycerol as a fuel for the body. Excess
adipose tissue leads to insulin resistance, thereby increasing the
risk of type 2 diabetes mellitus and cardiovascular disease (28).
Wang and Jones (29) proposed that decreased preadipocyte
proliferation and adipocyte lipogenesis are mechanisms of
Figure 5. Effect of capsaicin on caspase-3 activity and apoptotic protein
levels in 3T3-L1 preadipocytes. Cells were treated with 0−250 µM
capsaicin for 12 and 24 h. Results are the mean ± SD (n ) 3). Protein
levels were analyzed by Western blot analysis. The relative expression
of caspase-3, PARP, Bak, Bax, and Bcl-2 in 3T3-L1 preadipocytes was
quantified densitometrically using the software LabWorks 4.5 and calculated
according to the reference bands of ?-actin (mean, n ) 3); /p < 0.05
compared with the control.
Figure 6. Effect of capsaicin on the inhibition of intracellular triglycerides
(A) and GPDH activity (B) in 3T3-L1 adipocytes. Cells were treated with
0−250 µM capsaicin for 72 h. Results are the mean ± SD (n ) 3);
/p < 0.05 compared with the control.
Figure 7. Effect of capsaicin on protein expression of PPARγ, C/EBPR,
adiponectin, and leptin in 3T3-L1 adipocytes. Cells were treated with 0−100
µM capsaicin for 12 and 24 h. Protein levels were analyzed by Western
blot analysis. The relative expression of PPARγ, C/EBPR, adiponectin,
and leptin in 3T3-L1 adipocytes was quantified densitometrically using
the software LabWorks 4.5 and calculated according to the reference
bands of ?-actin.
J. Agric. Food Chem., Vol. 55, No. 5, 2007 Hsu and Yen
antiobesity. The 3T3-L1 preadipocytes are not cancer cells, but
the preadipocytes have been differentiated into mature adipo-
cytes. So, we focused on the effects of capsaicin on induction
of preadipocytic apoptosis and inhibition of adipocytic lipid
The goal of this research was to study the inhibition of
adipogenesis and adipocyte differentiation with a natural chemi-
cal. The results of the MTT and LDH assays clearly indicated
that capsaicin caused the inhibition of cell population growth
of 3T3-L1 preadipocytes (Figure 2, parts A and B). In the cell
apoptosis analysis by PI staining, treatment of 3T3-L1 preadi-
pocytes with capsaicin increased the induction of cell apoptosis
in a time- and dose-dependent manner (Figure 3A). Annexin
V-FITC binds to phosphatidylserine and can be used to detect
the early stages of apoptosis (30). Our data showed that the
treatment of 3T3-L1 preadipocytes with capsaicin increased the
apoptotic cell population in a dose-dependent manner (Figure
3B). Detection of the mitochondria membrane potential event
provided an early indication of the initiation of cellular
apoptosis. In general, changes in the membrane phosphatidyl
serine externalization are generally observed at a stage later than
the loss of mitochondria membrane potential (31). Mitochondria
play an essential role in cell death signal transduction such that
permeability transition pore opening and collapse of the ∆Ψm
results in the rapid release of caspase activators such as
cytochrome c into the cytoplasm (32). These results demon-
strated that the treatment of 3T3-L1 preadipocytes with capsaicin
increased the loss of mitochondria membrane potential in a time-
and dose-dependent manner (Figure 4).
In an attempt to reveal the molecular mechanisms underlying
capsaicin-induced apoptosis of 3T3-L1 preadipocytes, caspase-3
activity (Figure 5A) and the protein levels of various key
apoptosis-linked gene products including caspase-3, PARP, Bak,
Bax, and Bcl-2 (Figure 5B) were determined. The data also
showed that capsaicin-induced apoptosis is controlled through
caspase activation. Caspase-3 is one of the candidates for cell
death-inducing proteases that cleave PARP (33). Subsequent
Western blot analysis disclosed progressive proteolytic cleavage
of PARP in 3T3-L1 preadipocytes after treatment with capsaicin.
Moreover, capsaicin can modulate the process of apoptosis in
tumor cells through caspase activation, a decrease of Bcl-2
expression, and an increase in Bax expression (34). In the
present study, the data showed that the decreased level of
antiapoptotic Bcl-2 protein and increased level of proapoptotic
Bax and Bak proteins may play a key role in capsaicin-induced
apoptosis of 3T3-L1 preadipocytes. Yang et al. (35) indicated
that esculetin-mediated adipocyte apoptosis involves the mito-
Our data indicated that the exposure of 3T3-L1 adipocytes
to capsaicin caused a significant decrease (p < 0.05) in the
content of intracellular triglycerides and GPDH (Figure 6).
However, it did not affect the cell number of 3T3-L1 adipocytes.
One report has indicated that CLA treatment of rats does not
decrease adipocyte number (36). Mochizuki and Hasegawa (37)
demonstrated that when adipocytes were exposed to green tea
catechins, EGCG inhibited lipogenesis, while (+)-catechins
increased lipogenesis. Mori and Hasegawa (38) indicated that
powdered green tea inhibited insulin-induced lipogenesis and
increased the level of superoxide dismutase (SOD) activity in
3T3-L1 adipocytes. In the present study, the results indicated
that treatment with capsaicin markedly decreased PPARγ,
C/EBPR, and leptin protein expression in 3T3-L1 adipocytes
(Figure 7). The promoters of several adipogenic genes are
regulated by PPARγ and C/EBPR transcription factors (39).
Moreover, PPARγ target genes in adipose tissue are directly
implicated in lipogenic pathways (40). Adiponectin is an
adipocytokine that has been shown to have antiatherogenic, anti-
inflammatory, and antidiabetic roles (41). Leptin is a protein
hormone secreted mainly by adipose tissue, and it inhibits food
intake and stimulates thermogenesis (42). Jeon et al. (43)
indicated that red yeast rice extracts induced the down-regulation
of adipogenic transcription factors and gene expression in 3T3-
L1 adipocytes. Our data suggested that capsaicin decreased the
levels of PPARγ, C/EBPR, and leptin and then increased the
level of adiponectin in 3T3-L1 adipocytes.
Bioavailability is the extent to which a nutrient in a food
constituent can be absorbed and used by the body after ingestion.
The reported indicated that the intake of capsaicin in a typical
Indian or Thai diet was about 128 µg/kg human body weight
(44). The concentrations of capsaicin in the gastric fluid (1-3
L) would be equivalent to 8-25 µM for an adult human with
a body weight of 60 kg. However, data presented here indicated
that capsaicin efficiently induces apoptosis and inhibits adipo-
genesis in 3T3-L1 cells at concentrations below 50 µM. The
range of concentrations used in the present study was consistent
with those in another study on the effect of capsaicin in 3T3-
L1 cells (45).
In conclusion, the results of this study clearly showed that
capsaicin could inhibit the population growth and the induction
of apoptosis in 3T3-L1 preadipocytes. Capsaicin induced
apoptosis through the collapse of mitochondria membrane
potential, activation of caspase-3, Bax, and Bak, and then
cleavage of PARP and down-regulation of Bcl-2. Capsaicin also
inhibited lipid accumulation and the protein expression of
PPARγ, C/EBPR, and leptin, but induced up-regulation of
adiponectin in 3T3-L1 adipocytes. These results demonstrate
that capsaicin efficiently suppresses adipogenesis in 3T3-L1
preadipocytes and adipocytes.
Annexin V-FITC, annexin V-fluorescein isothiocyanate;
C/EBPR, CCAAT enhancer binding protein alpha; DEX,
dexamethasone; DMEM, Dulbecco’s modified Eagle’s medium;
DMSO, dimethyl sulfoxide; GPDH, glycerol-3-phosphate de-
hydrogenase; IBMX, 3-isobutyl-1-methylxanthine; IC50, 50%
growth inhibitory concentrations; INS, insulin; MTT, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; ∆Ψm,
mitochondria membrane potential; OROSM, oil red O-stained
material; PARP, poly(ADP-ribose) polymerase; PBS, phosphate-
buffered saline; PI, propidium iodide; PPARγ, peroxisome
proliferator-activated receptor-gamma; PVDF, polyvinyldif-
luoride; RNase, ribonuclease; TE, trypsin-EDTA.
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Received for review October 11, 2006. Revised December 29, 2006.
Accepted January 8, 2007. This research work was partially supported
by the Department of Health, Taiwan, ROC, under Grant DOH95-
J. Agric. Food Chem., Vol. 55, No. 5, 2007Hsu and Yen