Combined effects of genistein, quercetin, and resveratrol in human and 3T3-L1 adipocytes.
ABSTRACT The natural compounds genistein (G), quercetin (Q), and resveratrol (R) have been reported to each exhibit anti-adipogenic activities in adipocytes and antiproliferative and pro-apoptotic activities in several cell types. We studied the combined effects of G, Q, and R on adipogenesis and apoptosis in primary human adipocytes (HAs) and 3T3-L1 murine adipocyte (MAs). Combined treatment with 6.25 microM G, 12.5 microM Q, and 12.5 microM R during the 14-day differentiation period caused an enhanced inhibition of lipid accumulation in maturing HAs that was greater than the responses to individual compounds and to the calculated additive response. Glycerol 3-phosphate dehydrogenase activity, a marker of late adipocyte differentiation, was decreased markedly in HAs treated with the combination of G+Q+R. In addition, combined treatment with 50 microM G, 100 microM Q, and 100 microM R for 3 days decreased cell viability and induced apoptosis in early- and mid- phase maturing and lipid-filled mature HAs. In contrast, no compound alone induced apoptosis. Oil Red O stain and Hoechst 33342 stain were performed to confirm the effects on lipid accumulation and apoptosis, respectively. We also determined whether MAs responded to the combination treatment similarly to HAs. As in HAs, G+Q+R treatment decreased lipid accumulation in maturing MAs and increased apoptosis in pre- and lipid-filled mature MAs more than the responses to G, Q, and R when used separately. These results show that lower concentrations of combined treatments with several natural compounds may be useful for treatments for obesity through the suppression of adipogenesis and enhanced adipocyte apoptosis.
- SourceAvailable from: unam.mx[show abstract] [hide abstract]
ABSTRACT: Resveratrol (3,5,4'-trihydroxystilbene) is the parent compound of a family of molecules, including glucosides and polymers, existing in cis and trans configurations in a narrow range of spermatophytes of which vines, peanuts and pines are the prime representatives. Its synthesis from p-coumaroyl CoA and malonyl CoA is induced by stress, injury, infection or UV-irradiation, and it is classified as a phytoalexin anti-fungicide conferring disease resistance in the plant kingdom. In vitro, ex vivo and animal experiments have shown that it possesses many biological attributes that favour protection against atherosclerosis, including antioxidant activity, modulation of hepatic apolipoprotein and lipid synthesis, inhibition of platelet aggregation as well as the production of pro-atherogenic eicosanoids by human platelets and neutrophils. Red wine represents its main source in the human diet, and it has been proposed as a major constituent of the polyphenol fraction to which the health benefits of red wine consumption have been attributed. The past several years have witnessed intense research devoted to its measurement in wine and the factors likely to promote its enrichment in this beverage. Up to the present, conclusive evidence for its absorption by human subjectsin biologically significant amounts is lacking, and it is questionable (but not yetexcluded) that its powerful and beneficial in vitro activities are reproduced as a consequence of sustained moderate red wine consumption.Clinical Biochemistry 04/1997; 30(2):91-113. · 2.45 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Calorie restriction extends lifespan in organisms ranging from yeast to mammals. In yeast, the SIR2 gene mediates the life-extending effects of calorie restriction. Here we show that the mammalian SIR2 orthologue, Sirt1 (sirtuin 1), activates a critical component of calorie restriction in mammals; that is, fat mobilization in white adipocytes. Upon food withdrawal Sirt1 protein binds to and represses genes controlled by the fat regulator PPAR-gamma (peroxisome proliferator-activated receptor-gamma), including genes mediating fat storage. Sirt1 represses PPAR-gamma by docking with its cofactors NCoR (nuclear receptor co-repressor) and SMRT (silencing mediator of retinoid and thyroid hormone receptors). Mobilization of fatty acids from white adipocytes upon fasting is compromised in Sirt1+/- mice. Repression of PPAR-gamma by Sirt1 is also evident in 3T3-L1 adipocytes, where overexpression of Sirt1 attenuates adipogenesis, and RNA interference of Sirt1 enhances it. In differentiated fat cells, upregulation of Sirt1 triggers lipolysis and loss of fat. As a reduction in fat is sufficient to extend murine lifespan, our results provide a possible molecular pathway connecting calorie restriction to life extension in mammals.Nature 07/2004; 429(6993):771-6. · 38.60 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Anticarcinogenic effects of polyphenolic compounds in fruits and vegetables are well established. Although polyphenols naturally occur as combinations, little information is available regarding possible synergistic or antagonistic biochemical interactions between compounds. Identifying potential interactions between polyphenols may provide information regarding the efficiency of polyphenol-containing foods in cancer prevention. The objective of this study was to investigate the interactions of ellagic acid and quercetin with resveratrol, polyphenols which occur in muscadine grapes, with the hypothesis that the selected polyphenols would interact synergistically in the induction of apoptosis and reduction of cell growth in human leukemia cells (MOLT-4). To test this hypothesis, alterations in cell cycle kinetics, proliferation, and apoptosis (caspase-3 activity) were examined after incubation with ellagic acid, quercetin, and resveratrol as single compounds and in combination. Results showed a more than additive interaction for the combination of ellagic acid with resveratrol and furthermore, significant alterations in cell cycle kinetics induced by single compounds and combinations were observed. An isobolographic analysis was performed to assess the apparent synergistic interaction for the combinations of ellagic acid with resveratrol and quercetin with resveratrol in the induction of caspase 3 activity, confirming a synergistic interaction with a combination index of 0.64 for the combination of ellagic acid and resveratrol and 0.68 for quercetin and resveratrol. Results indicate that the anticarcinogenic potential of foods containing polyphenols may not be based on the effects of individual compounds, but may involve a synergistic enhancement of the anticancer effects.Cancer letters 03/2005; 218(2):141-51. · 4.86 Impact Factor
JOURNAL OF MEDICINAL FOOD
J Med Food 11 (4) 2008, 11:773–783
© Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition
PARK ET AL.
Combined Effects of Genistein, Quercetin, and Resveratrol in Human and 3T3-L1 Adipocytes
Hea Jin Park,1 Jeong-Yeh Yang,1 Suresh Ambati,1 Mary Anne Della-Fera,1 Dorothy B.
Hausman,2 Srujana Rayalam,1 and Clifton A. Baile1,2
Departments of 1Animal & Dairy Science and 2Foods and Nutrition, University of Georgia,
Manuscript received 14 March 2008. Revision accepted 13 April 2008.
Address reprint requests to: Clifton A. Baile, 444 Edgar L. Rhodes Center for Animal and Dairy
Science, University of Georgia, Athens, GA 30602-2771, E-mail: email@example.com
ABSTRACT The natural compounds genistein (G), quercetin (Q), and resveratrol (R) have been
reported to each exhibit anti-adipogenic activities in adipocytes and antiproliferative and pro-
apoptotic activities in several cell types. We studied the combined effects of G, Q, and R on
adipogenesis and apoptosis in primary human adipocytes (HAs) and murine 3T3-L1 adipocyte
(MAs). Combined treatment with 6.25 μM G, 12.5 μM Q, and 12.5 μM R during the 14-day
differentiation period caused an enhanced inhibition of lipid accumulation in maturing HAs that
was greater than the responses to individual compounds and to the calculated additive response.
Glycerol 3-phosphate dehydrogenase activity, a marker of late adipocyte differentiation, was
decreased markedly in HAs treated with the combination of G+Q+R. In addition, combined
treatment with 50 μM G, 100 μM Q, and 100 μM R for 3 days decreased cell viability and
induced apoptosis in early- and mid- phase maturing and lipid-filled mature HAs. In contrast, no
compound alone induced apoptosis. Oil Red O stain and Hoechst 33342 stain were performed to
confirm the effects on lipid accumulation and apoptosis, respectively. We also determined
whether MAs responded to the combination treatment similarly to HAs. As in HAs, G+Q+R
treatment decreased lipid accumulation in maturing MAs and increased apoptosis in pre- and
lipid-filled mature MAs more than the responses to G, Q, and R when used separately. These
results show that lower concentrations of combined treatments with several natural compounds
may be useful for treatments for obesity through the suppression of adipogenesis and enhanced
KEY WORDS: • adipogenesis • apoptosis • combined effects • genistein • phytochemicals •
quercetin • resveratrol
RUITS AND VEGETABLES are well known to have potential for reducing the incidence of
cancer and cardiovascular disease due to their broad range of phytochemicals, such as
flavonoids. Flavonoid intake is inversely correlated with mortality from cardiovascular disease
in population studies,1 and many flavonoids have also been studied for their effects on adipose
tissue in vivo and in vitro.2,3 Genistein (4,5,7-trihydroxyisoflavone) (G), the most abundant
isoflavone found in soybeans, has a structure similar to that of estrogen and has high affinity to
estrogen receptor-.4 It has been shown to decrease food intake, body weight, and fat pad weight
and induce apoptosis of adipose tissue in ovariectomized mice.5 G has also been reported to
inhibit lipid accumulation in 3T3-L1 adipocytes,2,6 to inhibit cell proliferation in cancer cells and
adipocytes,2,7 and to increase lipolysis in adipocytes.2,8 Quercetin (3,5,7,3′,4′-
pentahydroxyflavone) (Q), a flavonol found in often-consumed fruits and vegetables,9 has been
reported to inhibit glucose uptake10 and to increase lipolysis11 in rat adipocytes. Induction of
apoptosis has also been demonstrated by Q in 3T3-L1 adipocytes and cancer cells.12 Resveratrol
(trans-3,5,4'-trihydroxystilbene) (R), a phytoalexin found in various fruits,13 has been shown to
reduce the synthesis of lipids in rat liver14 and 3T3-L1 adipocytes.15 R has also been reported to
play a role in suppressing proliferation and inducing apoptosis in hematopoietic cells in vitro.16
Numerous studies have investigated the effects of individual natural compounds on cells
in an effort to determine underlying mechanisms of action. However, relatively few studies have
investigated the influence of combinations of natural compounds, although they occur naturally
as combinations in low concentrations. Nevertheless, some natural compounds have shown
synergistic or additive effects with other compounds.17 Yang et al.18 showed that trans-10,cis-12-
conjugated linoleic acid potentiated the effect of ajoene on apoptosis in 3T3-L1 adipocytes.
Mouria et al.19 demonstrated that R and Q additively activated caspase-3 in human pancreatic
carcinoma cells, and Mertens-Talcott et al.20 found that, in combination, R and Q synergistically
induced apoptosis in human leukemia cells. It is believed that these compounds interact with
different signal transduction pathways or stabilize each other.
There has been very limited study of human preadipocytes in primary cultures even
though the primary human adipocytes (HAs) are a preferred cell culture model for human obesity.
This paucity of human cell culture research is probably due to limited preadipocyte yields from
adipose tissue, as well as the amount of adipose tissue, because allowing proliferation of human
preadipocytes in culture inhibits subsequent differentiation.21 This is one reason why many
studies on adipocytes are conducted in murine cell lines, such as 3T3-L1 cells, and in animal
tissues. In this study, we used both primary HAs and mouse 3T3-L1 cells to test the effects of
three well-known phytochemicals, G, Q, and R, on adipogenesis and apoptosis in adipocytes.
MATERIALS AND METHODS
G (>99.0%) was purchased from Indofine Chemical Co. (Hillsborough, NJ). Q (97.0%)
and R (99.4%) were obtained from Chromadex Inc. (Santa Ana, CA). G, Q, and R were
dissolved in dimethyl sulfoxide (DMSO). AdipoRed™ Assay reagent was purchased from
Cambrex BioScience (Walkersville, MD). CellTiter Blue® Cell Viability Assay reagent and
CellTiter 96® Aqueous One Solution Cell Proliferation Assay were obtained from Promega
(Madison, WI). ApoStrand enzyme-linked immunosorbent assay apoptosis detection kits were
purchased from BIOMOL (Plymouth Meeting, PA). Oil Red O stain and Hoechst stain were
obtained from Sigma (St. Louis, MO).
Culturing of primary HAs. The cells were purchased as cryopreserved preadipocytes from
Zen-Bio, Inc. (Research Triangle Park, NC). The pooled cells were obtained from subcutaneous
adipose tissue of seven women between 27 and 51 years of age with a mean body mass index of
25.17 kg/m2 (range, 22.5–28.2 kg/m2) who were not diabetic and not smokers. The cells were
cultured according to the manufacturer’s instructions with slight modifications. In brief,
cryopreserved preadipocytes were passaged one time with preadipocyte medium (PM-1,
consisting of Dulbecco’s Modified Eagle’s Medium [DMEM]/Ham’s F-12 medium, HEPES,
fetal bovine serum [FBS], penicillin, streptomycin, and amphotericin B; Zen-Bio, Inc.), then
plated at 40,625 cells/cm2 with PM-1, and cultured until confluent. To induce differentiation,
PM-1 was replaced with differentiation medium (DM-2, Zen-Bio, Inc.) including biotin,
pantothenate, human insulin, dexamethasone, isobutylmethylxanthine, and peroxisome
proliferator-activated receptor-γ agonist (Day 0). After 7 days, DM-2 was removed, and cells
were incubated with adipocyte medium (AM-1, consisting of PM-1, biotin, pantothenate, human
insulin, and dexamethasone; Zen-Bio, Inc.) for an additional 7 days. On Day 14, the majority of
cells contained large lipid droplets and were designated lipid-filled HAs.
Culturing of 3T3-L1 murine adipocytes (MAs). 3T3-L1 mouse embryo fibroblasts were
obtained from American Type Culture Collection (Manassas, VA) and cultured as described
elsewhere.18 In brief, cells were cultured in DMEM (GIBCO, Grand Island, NY) with 10%
bovine calf serum until confluent and stimulated to differentiate with DMEM containing 10%
FBS, 167 nM insulin, 0.5 M isobutylmethylxanthine, and 1 M dexamethasone for 2 days.
Cells were then maintained in 10% FBS/DMEM with insulin for another 2 days, followed by
culturing with 10% FBS/DMEM for an additional 4 days.
HAs and MAs were maintained at 37°C in a humidified 5% CO2 atmosphere.
Quantification of lipid content
Lipid content was quantified using AdipoRed assay reagent according to the
manufacturer’s instructions. In HA treatments (6.25 or 12.5 μM G, 12.5 or 25 μM Q, and 12.5 or
25 μM R and their combinations) were added with DM-2 and AM-1 from Days 0 to 14, and
0.3% DMSO was used as a control. For the time course experiment, treatments (6.25 μM G, 12.5
μM Q, and 12.5 μM R and their combination) were added with DM-2 for Days 0–7 or added with
AM-1 for Days 7–14. In MAs, treatments (25 μM G, 25 μM Q, and 12.5 μM R and their
combination) were added from Days 0 to 6. Medium with treatment was changed every 2–3 days.
On Day 14 for HAs or on Day 6 for MAs, intracellular lipid content was measured by AdipoRed
assay. Cells were incubated with 200 μL of phosphate-buffered saline and 5 μL of AdipoRed
reagent for 10 minutes at room temperature, and fluorescent signal was measured with excitation
at 485 nm and emission at 572 nm.
Cell viability assay
Primary HAs. HAs were treated with either test compounds (6.25 μM G, 12.5 μM Q, and
12.5 μM R and their combination) or vehicle (0.3% DMSO) during the adipogenesis period as
described above. In addition, early- (Day 0) and mid-phase (Day 7) maturing HAs and lipid-
filled HAs (Day 14) were incubated with test compounds (50 μM G, 100 μM Q, and 100 μM R
and their combination) for 3 days in DM-2 (Days 0–3), AM-1 (Days 7–9) and FBS-added BM-1
(BM-1; DMEM/Ham’s F-12 medium, HEPES, FBS, biotin, pantothenate, penicillin,
streptomycin, and amphotericin B; Zen-Bio, Inc.) (Days 14–16), respectively. On an assay day,
the treatment medium was replaced with 100 µL of fresh medium and 20 µL of CellTiter Blue
cell viability reagents. Cells were then incubated in the dark for 1 hour at 37°C, and the
fluorescent signal was measured at an excitation wavelength of 560 nm and an emission
wavelength of 590 nm to determine the resorufin concentration, which is proportional to the
number of live cells.
3T3-L1 MAs. MAs were treated with either test compounds (25 μM G, 25 μM Q, and 12.5
μM R and their combination) or vehicle (0.3% DMSO) during the adipogenesis period as
described above. In addition, pre- and lipid-filled MAs were incubated with test compounds (50
μM G, 50 μM Q, and 50 μM R and their combination) for 2 days. On the assay day, the
treatment medium was replaced with 100 µL of fresh medium and 20 µL of MTS (3-(4,5-
Cells were then incubated in dark for 1 hour at 37°C, and the absorbance was measured at 490
nm to determine the formazan concentration, which is proportional to the number of live cells.
Oil Red O stain
Cells were treated with test compounds (6.25 μM G, 12.5 μM Q, and 12.5 μM R and their
combination for HAs, 25 μM G, 25 μM Q, and 12.5 μM R and their combination for MAs)
during the adipogenesis period as described above. Cells were fixed with Baker’s formalin (10
mL of 37% formaldehyde, 10 mL of a 10% calcium chloride solution, and 80 mL of distilled
water) for 30 minutes at room temperature and then stained with Oil Red O and hematoxylin.22
After staining, cells were mounted with glycerol gelatin, and the images were captured using
ImagePro software (MediaCybernetics, Silver Spring, MD).
Glycerol 3-phosphate dehydrogenase (GPDH) activity
Adipocytes were treated with test compounds (6.25 μM G, 12.5 μM Q, and 12.5 μM R
and their combination for HAs; 25 μM G, 25 μM Q, and 12.5 μM R and their combination for
MA) during the adipogenesis period as described above. On Day 14, cells were harvested with
0.5 mL of ice-cold buffer containing 0.28 M sucrose, 5 M Tris, 1 M EDTA, and 0.002% β-
mercaptoethanol. The homogenate was then sonicated and centrifuged at 10,000 g for 10
minutes at 4°C. The supernatants were used for GPDH assays according to the procedure of
Wise and Green.23 Activities are expressed in mU/mg of protein (1 mU being equal to the
oxidation of 1 nmol of NADH2/minute).
ApoStrand enzyme-linked immunosorbent assay apoptosis detection kits (BIOMOL
(Plymouth Meeting, PA) were used for measuring the extent of apoptosis. HAs and MAs were
incubated with test compounds (50 μM G, 100 μM Q, and 100 μM R and their combination for
HAs, 50 μM G, 50 μM Q, and 50 μM R and their combination for MAs) for indicated periods.
Cells were then fixed and assayed according to the manufacturer’s instructions. This assay is
based on the selective denaturation of DNA in apoptotic cells by formamide, which reflects
changes in chromatin associated with apoptosis. The absorbance was read at 405 nm.
Hoechst 33342 stain
Lipid-filled HAs were incubated with test compounds (50 μM G + 100 μM Q + 100 μM
R) for 1 day. Cells were then fixed with Baker’s formalin and stained with 10 M Hoechst dye
for 15 minutes in the dark. After staining, cells were observed by fluorescence microscopy
(MediaCybernetics). Cells with bright blue fragmented nuclei showing condensation of
chromatin were identified as apoptotic cells.
One-way or two-way analysis of variance, where appropriate, was used to determine the
significance of treatment or treatment and time effects (GLM procedure, Statistica, version 6.1;
StatSoft®, Tulsa, OK, USA). 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 ± standard error values.
Combined treatment with G, Q, and R inhibited lipid accumulation in primary HAs
HAs were treated with test compounds during the differentiation period. Preliminary
tests were carried out to determine the best concentrations to use in order to detect potential
enhanced combination treatment effects (data not shown). The concentrations selected for this
experiment were 6.25 and 12.5 M G, 12.5 and 25 M Q, and 12.5 and 25 M R.
Lipid accumulation was decreased with 12.5 μM G (47.9 ± 1.5%, P < .01), 25 μM R
(22.1 ± 1.5%, P < .01), and 25 μM Q (16.7 ± 0.5%, P < .01). However, lower concentrations of
R had no effect, and 12.5 μM Q increased lipid content by 17.5 ± 1.4% (P 0 01). Only 6.25 μM
G decreased lipid content by 18.8 ± 1.1% (P < .01). Combined treatment with 6.25 μM G + 12.5
μM Q + 12.5 μM R (G+Q+R) suppressed lipid accumulation by 92.4 ± 0.4% (P < .01), showing
more than the calculated additive effect during the differentiation period (Fig. 1A). Similar
results were observed using Oil Red O stain to visualize lipid accumulation in cells (Fig. 1B).
Cells were treated with 6.25 μM G + 12.5 μM Q + 12.5 μM R (G+Q+R) for different
periods during the differentiation process. There was no significant difference in lipid content of
cells treated with G+Q+R during Days 0–7 and Days 0–14 (Fig. 2A). In addition, G+Q+R
treatment during Days 0–14 decreased cell viability by 29.3 ± 0.8% (P < .01) (Fig. 2B).
GPDH activity in undifferentiated cells was only 5.0 ± 0.2% of that in lipid-filled HAs
(Fig. 3). GPDH activity was not influenced by either 6.25 M G or 12.5 M Q, while 12.5 M R
decreased GPDH activity by 31.1 ± 3.0% (P < .01). G+Q+R treatment decreased GPDH activity
by 52.2 ± 3.8% (P < .01).
Combined treatment with G, Q, and R decreased cell viability and increased apoptosis in
Following preliminary testing, cell viability and apoptosis were assessed with treatments
of 50 M G, 100 M Q, and 100 M R. Treatments were added for 3 days in early- and mid-
phase maturing and lipid-filled HAs. In early-phase maturing HAs (Fig. 4A), G and R decreased
viability by 13.1 ± 1.5% (P < .01) and 13.8 ± 0.8% (P < .01), respectively, while Q had no effect.
In mid-phase maturing HAs (Fig. 4B), G and Q decreased viability by 9.4 ± 0.9% (P < .01) and
19.4 ± 0.9% (P < .01), respectively, while R had no effect. In lipid-filled HAs, Q and R
decreased viability by 9.1 ± 2.2% (P < .01) and 9.0 ± 2.5% (P < .01), respectively, while G had
no effect (Fig. 4C). However, combined G+Q+R reduced viability by 39.4 ± 0.8% (P < .01) in
early-phase maturing HAs, 58.3 ± 1.4% (P < .01) in mid-phase maturing HAs, and by 55.4 ±
1.9% (P < .01) in lipid-filled HAs.
None of the individual compound treatments (50 M G, 100 M Q, and 100 M R)
induced apoptosis in these stages of HAs (Fig. 5A–C). The combination treatment, however,
increased apoptosis by 46.5 ± 6.6% (P < .01), 90.6 ± 20.6% (P < .01), and 64.2 ± 15.1% (P
< .01) in early- and mid-phase maturing and lipid-filled HAs, respectively. Using Hoechst 33342
stain, apoptotic cells treated with G+Q+R were identified, as shown in Fig. 5D, with bright blue
fragmented nuclei showing condensation of chromatin.
Combined treatment with G, Q, and R inhibited lipid accumulation and induced apoptosis in
MAs were incubated with 25 μM G, 25 μM Q, and 12.5 μM R during the differentiation
period (Fig. 6A). The selection of concentrations to test was based on preliminary tests (data not
shown). When MAs were treated with G, Q, and R individually, lipid accumulation was
decreased by 16.8 ± 4.0% (P < .01), 20.3 ± 4.7% (P < .01), and 17.4 ± 2.6% (P < .01),
respectively. When the three compounds were added in combination, lipid accumulation was
decreased by 80.3 ± 2.2% (P < .01). Cell viability was decreased with 25 M G (12.6 ± 1.2%, P
< .01) and G+Q+R (13.3 ± 1.6%, P < .01) (Fig. 6B and C).
Postconfluent preadipocytes and lipid-filled MAs were tested with 50 M G, 50 M Q,
and 50 M R to determine the effect on apoptosis (Fig. 7). In pre-MAs, G+Q+R decreased
viability by 45.8 ± 1.5% (P < .01), while G, Q, and R individually decreased viability only 2.6 ±
3.7% (not significant), 9.1 ± 0.9% (P < .05), and 17.7 ± 1.5 (P < .01), respectively. In lipid-filled
MAs, G+Q+R decreased viability 80.8 ± 1.5% (P < .01); however, of the three components
tested alone at the same concentrations, only R decreased viability, by 10.2 ± 4.2% (P < .01).
The decreased viability at these concentrations was associated with increased apoptosis: G+Q+R
increased apoptosis by 121.8 ± 30.0% (P < .01) in pre-MAs and 432.2 ± 29.8% (P < .01) in
lipid-filled MAs, while G, Q, and R as individual treatments had no significant effect on either
The combined effect with G, Q and R on adipogenesis and apoptosis was examined in
HAs as well as in MAs in the current study. G has been shown in 3T3-L1 adipocytes to inhibit
lipid accumulation in part by decreasing the expression of peroxisome proliferator-activated
receptor-γ and CCAAT/enhancer binding protein 2,24 and activating AMP-activated protein
kinase.6 Resveratrol has also been reported to inhibit adipogenesis by repressing peroxisome
proliferator-activated receptor-γ activity with activation of Sirt 1.15 In our study with HAs, 12.5
μM G, 25 μM R, and 25 μM Q decreased lipid accumulation significantly. However, lower
concentrations of R had no effect, and 12.5 μM Q increased lipid content. Only 6.25 μM G
decreased lipid content in HAs. Interestingly, when lower concentrations of G, Q, and R (6.25
μM G + 12.5 μM Q + 12.5 μM R) were combined, lipid accumulation was almost completely
inhibited, showing more than the calculated additive effect during the differentiation period.
To determine the time during which cells were most sensitive to the inhibition of
adipogenesis, cells were treated for different periods during the differentiation process.
Combined 6.25 μM G + 12.5 μM Q + 12.5 μM R (G+Q+R) inhibited adipogenesis at all periods,
but the time period of Days 0–7 proved to be critical for the anti-adipogenic effect. G+Q+R for
Days 0–7 completely inhibited adipocyte differentiation, but when cells were incubated with
treatment for Days 7–14, the inhibitory effect was far less pronounced. In addition, the
decreased lipid accumulation with G+Q+R was due not only to inhibited lipid accumulation but
also to decreased cell viability.
The enhanced effect on lipid accumulation was also partly due to an effect on GPDH
activity. GPDH is an enzyme that catalyzes the reversible reaction between dihydroxyacetone
phosphate and glycerol 3-phosphate with NAD as a coenzyme. It is known that GPDH activity
increases during differentiation of progenitor cells to adipocytes, and it has been used as an index
for monitoring triglyceride synthesis.23 In our study, G+Q+R treatment markedly decreased
GPDH activity. Although this appeared to be only an additive effect, it was substantial.
The number of adipocytes in adipose tissue can be decreased by inducing apoptosis as
well as inhibiting adipogenesis.25 In this study, we used 50 μM G, 100 μM Q, and 100 μM R,
none of which individually induced apoptosis in any cell stage of HAs. The combination
treatment, however, increased apoptosis by 46.5%, 90.6%, and 64.2% in early- and mid-phase
maturing HAs and lipid-filled HAs, respectively. G has been shown to inhibit cell proliferation
in 3T3-L1 adipocytes2 and tumor cells8,26 and to induce apoptosis in adipose tissue of
ovariectomized mice and in 3T3-L1 adipocytes.5 Q-induced apoptosis is accompanied by
inhibition of c-Jun N-terminal kinase activation27 and release of cytochrome c by caspase-3
activation in cancer cells.19 Hsu and Yen12 also showed that Q-induced apoptosis in 3T3-L1
adipocytes is accompanied by decreased mitochondrial membrane potential, increased caspase-3
activity, down-regulated poly(ADP-ribose) polymerase and Bcl-2, and activated Bax and Bak
proteins. R also induces apoptosis with release of cytochrome c in cancer cells.19,28 Interestingly,
Mouria et al.19 reported that in human leukemia cells Q and R showed a greater effect on
cytochrome c release and caspase-3 activity than the expected additive response in human
leukemia cells. In addition, we have recently reported enhanced effects of G plus R in inhibiting
adipogenesis and inducing apoptosis in 3T3-L1 adipocytes.29 In that study the combination of
genistein plus resveratrol at either 50 M each or 100 M each increased apoptosis more than the
additive responses to the individual compounds. Interestingly, the combination increased c-Jun
N-terminal kinase phosphorylation and decreased extracellular signal-regulated kinase 1/2
phosphorylation, whereas the compounds tested individually had no effect. Furthermore,
adipogenesis was inhibited more by the combination of G plus R at 25 M each than by the
additive responses to the individual compounds. Although the individual compounds had no
effect on peroxisome proliferator-activated receptor-γ and CCAAT/enhancer binding protein
expression, the combination of G plus R suppressed the expression of both.
In a separate study combinations of R and G were tested for effects on adipogenesis and
apoptosis in 3T3-L1 adipocytes.30 That study also showed that the combinations caused greater
inhibition of adipogenesis and induction of apoptosis than would have been predicted based on
the additive effects of the individual compounds.
In the current study we selected concentrations of G, Q, and R that were individually
ineffective at inducing apoptosis but in combination caused a significant increase in apoptosis.
In previous studies in which we found enhanced effects of combinations of compounds on
adipogenesis or apoptosis, we also demonstrated enhanced activity in altering different signal
transduction pathways. For example, Rayalam et al.31 reported that the combination of 1,25-
dihydroxy-vitamin D3 and guggulsterone was more potent than either of the individual
compounds in decreasing adipogenesis, presumably because of an effect of the combination on
altering the expression of 1,25-dihydroxy-vitamin D3 receptor and farnesoid X receptors.
Likewise, similar effects were noticed with 1,25-dihydroxy-vitamin D3 plus G.32 In that study
we observed that although G showed no effect on 1,25-dihydroxy-vitamin D3 receptor
expression during adipogenesis, the combination of vitamin D and G caused a potentiated
increase in the expression of 1,25-dihydroxy-vitamin D3 receptor, leading to decreased
adipogenesis. In addition, trans-10, cis-12 conjugated linoleic acid potentiates the effect of
ajoene on apoptosis through an increase in the phosphorylation of c-Jun N-terminal kinase, Bax
expression, reactive oxygen species production, and a greater release of mitochondrial proteins
(cytochrome c, apoptosis-inducing factor).18 Based on our previous reports and findings, we
therefore propose that the enhanced effects observed in the current study might be a result of
interaction of different signaling pathways.
We also determined whether the effect of these compounds on MAs was similar to that
on HAs. Whereas the primary HA culture includes both preadipocytes and fibroblast-like cells,
the 3T3-L1 cell line provides a homogeneous population with virtually all cells being at the same
stage. Owing to their convenience and tractability, 3T3-L1 cells have been used widely to study
preadipocyte biology.33 In MAs in this study, 25 μM G, 25 μM Q, and 12.5 μM R were tested
during the adipogenic period. The selection of concentrations to test was based on preliminary
testing, and the reason why different concentrations are required at the different stages and in the
different cell types is probably primarily due to the differences in incubation periods used. When
cells were treated with G, Q, and R individually, they decreased lipid accumulation by 16.8%,
20.3%, and 17.4%, respectively. When the three compounds were added in combination, lipid
accumulation was decreased by 80.3%, which is greater than the additive effect of individual
compounds. In addition to the effect on lipid accumulation, 25 M G and G+Q+R decreased cell
viability by 12.6% and 13.3%, respectively. In HAs, G+Q+R caused decreased viability as well
as decreased lipid accumulation compared to individual treatments. However, in MAs,