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3 Department of Animal and Dairy Science and 4 Department of Foods and Nutrition, University of Georgia, Athens, GA 30602-2771
* To whom correspondence should be addressed. E-mail: cbaile{at}uga.edu.
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ABSTRACT |
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 TOP ABSTRACT IntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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and CCAAT/enhancer binding protein-alpha were downregulated after treatment with G + R, but no effect was observed with individual compounds. These results indicate that G and R in combination produce enhanced effects on inhibiting adipogenesis, inducing apoptosis, and promoting lipolysis in 3T3-L1 adipocytes. Thus, the combination of G and R is more potent in exerting antiobesity effects than the individual compounds.
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Introduction |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Relatively little research exists on the effects of phytoestrogens on adipocytes. Genistein (G),5 a soy isoflavone, was shown to decrease food intake, body weight, and fat pad weight in ovariectomized female mice (5,6). In adipocytes, G was shown to inhibit cell proliferation and increase lipolysis (7). In addition to estrogenic effects, G has effects on protein tyrosine kinases, apoptosis, cell proliferation, and angiogenesis (8–10) and can potentially affect adipose tissue through these mechanisms. G was also implicated in cancer control, primarily because of its strong antiproliferative and apoptotic potential (11). Resveratrol (R; 3,5,4'-trihydroxystilbene), a naturally occurring phytoalexin found in red wines and grape juice, was shown to reduce the synthesis of lipids in rat liver (12) and 3T3-L1 adipocytes (13), inhibit the synthesis of eicosanoids in rat leukocytes (14), interfere in arachidonate metabolism (15), and inhibit the activity of some protein kinases (16). R decreased proliferation and induced apoptosis and cell cycle arrest in various cell lines (17–19). Considering the antiadipogenic and lipolytic effects of G and R in murine adipocytes, coupled with their antiproliferative activity in a number of cell lines, we hypothesized that G and R may act synergistically to inhibit the signals that promote adipogenesis and decrease adipose tissue mass by apoptosis.
Interaction among the members of the C/EBP and PPAR families plays an important role in the adipogenesis process. CCAAT/enhancer binding protein-beta (C/EBPß) is expressed immediately after the induction of differentiation, and then PPAR and CCAAT/enhancer binding protein-alpha (C/EBP
) act synergistically to promote adipogenesis (20–22). Harmon et al. (23,24) showed that G inhibited the expression of PPAR and C/EBP in 3T3-L1 cells. However, the effect of R on PPAR and C/EBP expression is not known. In this study, we predicted that G and R would inhibit adipogenesis by modulating the expression of C/EBP and PPAR.
Given that phytoestrogens inhibit proliferation of several cell lines (25,26), we investigated the combination effect of G and R on adipocyte apoptosis. Mitogen-activated protein kinase (MAPK) pathways regulate diverse cellular activities, including cell survival, apoptosis, and differentiation. The MAPK pathways consist of 3 parallel kinase modules, that is, the extracellular signal-regulating kinase (ERK1/2), the Jun-N-terminal kinase (JNK), and the p38 MAPK pathways. In general, JNK and p38 MAPK activation is associated with apoptosis induction (27), whereas ERK1/2 are preferentially activated by phorbol esters (28) and are cytoprotective (27). R downregulated MAPK/JNK/p38 in the vasculature (29). G was shown to decrease ERK1/2 phosphorylation in various cell lines (30). In this study, we predicted that G and R would induce apoptosis by modulating ERK1/2 and JNK pathways.
Our objective was to examine the possibility of interaction between G and R, resulting in enhanced inhibition of adipogenesis and induction of apoptosis in 3T3-L1 adipocytes.
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Materials and Methods |
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Cell viability and apoptosis assays. Tests were performed in 96-well plates. For mature adipocytes, cells were seeded (5000 cells/well) and grown to maturation as described above. For preadipocytes, cells were seeded (2500 cells/well) and assay performed 3 d after seeding. Preadipocytes or mature adipocytes were incubated with either 0.2% dimethyl sulfoxide (DMSO) or test compounds for 24 and 48 h. Cell viability assay was performed per the manufacturer's instructions. The absorbance was measured at 490 nm in a plate reader (µQuant, Bio-Tek Instruments) to determine the formazan concentration, which is proportional to the number of live cells. For measuring the extent of apoptosis, ApoStrand ELISA apoptosis detection kit was used. Cells were grown in 96-well plates, treated with test compounds for 24 and 48 h, and assayed as per the manufacturer's instructions. The assay selectively detects single-stranded DNA, which occurs in apoptotic cells but not in necrotic cells or cells with DNA breaks in the absence of apoptosis (32). Assays were performed at least 2 times with 6 replicates for each treatment.
Quantification of lipid content. Lipid content was quantified using commercially available AdipoRed assay reagent. In brief, postconfluent preadipocytes grown in 96-well plates were incubated with 0.2% DMSO or test compounds during the adipogenic phase, and on d 6, cells were assayed for lipid content according to the manufacturer's instructions. The experiments were performed with at least 6 replicates per treatment and repeated 3 times.
Lipolysis assay. To determine the extent of lipolysis induced by test compounds, mature adipocytes were treated with either 0.2% DMSO or test compounds for 5 h, and the free glycerol released was assayed by using a Lipolysis assay kit for 3T3-L1 adipocytes (Zen-Bio) and following the manufacturer's instructions. The experiment was repeated 2 times with at least 4 replicates.
Western blot analysis. Mature adipocytes were treated with 100 µmol/L each of G and R as individual compounds and in combination for 3 h. Control cells were treated with 0.2% DMSO. Likewise, maturing preadipocytes were treated with 25 µmol/L each of G and R alone and in combination for 6 d. Whole cell extracts were prepared as described elsewhere (33). The protein concentration was determined by bicinchoninic acid assay with bovine serum albumin as the standard. Western blot analysis was performed using the commercial NUPAGE system (Novex/Invitrogen), where a lithium dodecyl sulfate 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 previously described (34).
Quantitative analysis of Western blot data. Measurement of signal intensity on polyvinylidene fluoride membranes after Western blotting with various antibodies was performed using a FluorChem densitomer with the AlphaEaseFC image processing and analysis software (Alpha Innotech). For statistical analysis, all data were expressed as integrated density values, which were calculated as the density values of the specific protein bands/ß-actin density values and expressed as a percentage of the control. All figures showing quantitative analysis include data from at least 3 independent experiments.
Reagents.
PBS and DMEM were purchased from GIBCO (BRL Life Technologies). ApoStrand ELISA apoptosis detection kit was purchased from BIOMOL. The viability assay kit (CellTiter 96 Aqueous one solution cell proliferation assay) was purchased from Promega. R was from Sigma. AdipoRed Assay Reagent was from Cambrex BioScience. G (99% pure) was purchased from Indofine Chemical Company. Antibodies specific for ß-actin, C/EBP, C/EBPß, and PPAR were purchased from Santa Cruz Biotechnology. Antibodies for phospho-JNK (Thr183/Tyr185), total JNK, phospho-ERK1/2 (Thr202/Tyr204), and total ERK1/2 were from Cell Signaling Technology.
Statistical analysis. ANOVA (GLM procedure, Statistica, version 6.1; StatSoft) was used to determine the significance of treatment and time effects and interactions (time vs. treatment). Fisher post-hoc least significant difference test was used to determine the significance of differences among means. In some cases, to estimate differences among the combined treatments and a hypothetical additive treatment response, a sum of the individual treatment effects for each replicate was calculated, and these numbers were included in the ANOVA. Statistically significant differences are defined at the 95% confidence interval. Data shown are means ± SE.
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Results |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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38.8 ± 4.4% (P < 0.001). Incubation of mature adipocytes for 3 h with G100 + R100 decreased ERK1/2 phosphorylation by 48 ± 3.4% (P < 0.001; Fig. 1B). R and G alone had no effect.
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Effects on PPAR, C/EBP, and C/EBPß expression.
To determine whether the decrease in lipid accumulation with G and R combinations was related to C/EBPß, C/EBP, and PPAR expression levels, whole cell lysates were extracted after treatment, as described previously, and subjected to Western blotting using anti-C/EBPß, anti-C/EBP, anti-PPAR, and anti-ß-actin antibodies. Quantitative analysis revealed that neither G25 nor R25 altered the expression levels of C/EBP and PPAR, whereas the combination (G25 + R25) decreased the expression levels of C/EBP by 56 ± 5.1% (P < 0.001; Fig. 3B) and PPAR by 48 ± 4.4% (P < 0.05; Fig. 3C), respectively. However, none of the treatments affected C/EBPß expression levels.
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Discussion |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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The relationship among dietary flavonoids and weight loss has not been explored adequately. However, in vivo studies suggest that isoflavones may be useful in the treatment of obesity. Isoflavone-rich diets improved lipid metabolism and had antidiabetic effects in obese rats (36). G, an isoflavone, was also shown to have direct effects on lipid metabolism in the liver and adipose tissue, decreasing triglycerides while increasing FFA in serum (37). Consistent with these results, in our study, G decreased lipid accumulation by 18% in maturing 3T3-L1 adipocytes, even at concentrations as low as 25 µmol/L. It was already demonstrated that apoptosis was a contributing factor to G's reducing effect on body weight (5), and in our study, G100 induced apoptosis by 50% more than the control. R also inhibited adipogenesis by 45% at 25 µmol/L, as shown previously (13). In addition, R induced apoptosis in mature adipocytes. Both of these flavonoids were effective by themselves at higher concentrations in inducing apoptosis, but not at 50 µmol/L. However, the effect of the combination (G50 + R50) in inducing apoptosis was not different from either G100 or R100 alone, although it was different from the calculated additive response (G50R50; Table 2). Likewise, the G100 + R100 combination had a greater effect than the calculated additive response (G100R100). Thus, based on limited dose testing, it is difficult to know whether the apoptotic effect was synergistic or additive. In contrast, G25 + R25 showed a much greater response in inhibiting adipogenesis than either G50 or R50, indicating that the combination effect is more than additive.
To elucidate the mechanism of apoptosis induced by G and R, we studied ERK1/2 and JNK expression. We found that G and R individually did not significantly alter ERK1/2 or JNK levels. However, in combination, they decreased ERK1/2 phosphorylation by 50% and increased JNK phosphorylation by 40%. ERK1/2 activation, in general, is considered to be cytoprotective, and JNK activation was shown to be associated with apoptosis induction (27). Though ERK1/2 activation results in cell proliferation (38), a few studies showed that, depending on the cell type, ERK1/2 activation may also result in cell death (39,40). Our finding that G did not activate JNK is not in agreement with the finding that G increased the activity of the JNK pathway in A431 cells (41), but it is in agreement with the finding that G did not activate JNK in MCF-10F cells (42). Inconsistent with our results, G inactivated ERK1/2 in MCF-10F cells (42). Similarly, R inhibited ERK1/2 signaling in A431 cells, which is not in agreement with our findings (43). R was shown to both activate and inhibit the JNK pathway (44,45). Cell-specific differences in the control of MAPK activity may contribute to these differences in response to G and R.
The adipogenesis process includes alteration of cell shape, growth arrest, and clonal expansion, leading to a complex sequence of changes in gene expression and lipid storage (46). C/EBPß is expressed in the first 2 d of differentiation, which corresponds to the period of mitotic clonal expansion (21). C/EBPß is one of the first transcription factors induced during the adipocyte differentiation, and it further mediates the expression of PPAR (47) and C/EBP (20–22). In this study, we did not observe changes in C/EBPß expression. Because the whole cell lysates from the treated cells were collected on d 6 after induction, we did not expect to see altered expression levels of C/EBPß because it is an early adipogenic marker. However, G and R in combination blocked differentiation by significantly suppressing the upregulation of both PPAR and C/EBP, correlating with results from the AdipoRed assay.
In addition to antiadipogenic effects, we also investigated the effects of G and R on lipolysis. G100 for 24 h induced a 6-fold greater release of glycerol into the culture medium than did the control in 3T3-L1 cells (23). In a different study, upregulation of Sirt1 by R was shown to trigger lipolysis in 3T3-L1 cells (13). These results are not in agreement with our findings that neither G25 nor R25 increased lipolysis. Differences in dose and incubation periods might contribute to the varied responses of these compounds. However, the combination of G and R significantly increased lipolysis, indicating that the antiadipogenic effect of this combination is at least partially mediated via enhancement of lipolysis.
The polyphenolic compounds present in fruits and vegetables regulate cell proliferation and induce apoptosis (48). R and quercetin, a flavonoid, synergistically induced apoptosis in human leukemia cells (49). Similarly, G and thearubigins (a flavone obtained from black tea) synergistically inhibited growth of prostate tumor cells. In this study, we showed that G and R synergistically inhibited adipogenesis and induced apoptosis in 3T3-L1 adipocytes. Although results from in vitro experiments cannot be directly extrapolated to clinical effects, these studies may help in elucidating various molecular pathways involved in the overall disease process of obesity. Moreover, a dose of 150 mg/kg G administered to ovariectomized female mice caused weight loss (5,6) and adipose tissue apoptosis (5), whereas 100 µmol/L G was the minimum concentration required to demonstrate a significant increase in apoptosis of 3T3-L1 adipocytes in vitro after 24 h (5). This is interesting because a dose of 150 mg/kg G in mice resulted in a plasma G concentration of 3.8 ± 0.4 µmol/L (6). Thus, these studies reflect the difficulty in making predictions about relationships among concentrations of agents that are shown to be effective in vitro under somewhat artificial conditions and effective plasma levels. To summarize, we demonstrated that G and R at tested concentrations are not very effective as individual compounds, but in combination, they are more capable of inducing apoptosis and decreasing lipid accumulation in adipocytes.
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FOOTNOTES |
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2 Author disclosures: S. Rayalam, J.-Y. Yang, H. J. Park, S. Ambati, no conflicts of interest; C. A. Baile and M. A. Della-Fera are investors in and serve on the Board of Directors for AptoTec Inc.
5 Abbreviations used: C/EBP, CCAAT/enhancer binding protein-alpha; C/EBPß, CCAAT/enhancer binding protein-beta; DMSO, dimethyl sulfoxide; ERK1/2, extracellular signal-regulating kinase; FBS, fetal bovine serum; G, genistein; G25, G50, G100, G at 25, 50, and 100 µmol/L; JNK, Jun-N-terminal kinase; MAPK, mitogen-activated protein kinase; R, resveratrol; R25, R50, R100, R at 25, 50, and 100 µmol/L.
Manuscript received 28 June 2007. Initial review completed 3 August 2007. Revision accepted 19 September 2007.
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