Increased visceral fat mass is associated with the development of metabolic disorders, including insulin resistance, dyslipidemia, hypertension, atherosclerosis, and inflammation [1
]. Obesity is considered as the main cause of metabolic disorders-related diseases, such as cardiovascular disease, hypertension, hyperlipidemia, type 2 diabetes, and cancer [2
]. Thus, the prevention of abnormal adipogenesis could be an efficient strategy for preventing the development of metabolic disorder-related diseases [3
Curcumin, a yellow phenolic component of turmeric, is derived from the rhizome of Curcuma longa L
. and has antioxidant [5
], anti-inflammation [6
], and anti-cancer properties [8
]. It also has several ameliorating effects on metabolic disorders. For example, dietary supplementation with curcumin significantly reduces obesity, inflammation, and diabetes in obese animal models [10
]. Curcumin also inhibits TNFα-stimulated inflammatory cytokine expression in adipocytes [10
] and suppresses preadipocyte differentiation in vitro [13
]. Supplementation with curcumin significantly reduces the high-fat diet-induced increase in body weight gain and adiposity in mice [13
]. A similar inhibition of differentiation of 3T3-L1 adipocytes was reported by Lee et al. [15
], who also found that curcumin can induce apoptosis of MCF-7 breast cancer cells; however, the apoptotic effect of curcumin on preadipocytes was not investigated. In addition, although curcumin is known to have an anti-obesity effect [10
], little is known about the underlying mechanisms of inhibition of adipogenesis.
The adipogenic process is divided into different stages of growth arrest, mitotic clonal expansion (MCE), and differentiation. Retinoblastoma protein (Rb), a tumor suppressor protein, plays an important role in the initial step of adipogenesis. Adipogenic hormones, such as isobutylmethylxanthine (IBMX), dexamethasone, and insulin, induce Rb phosphorylation through the cyclin-dependent kinase pathway, resulting in dissociation of the Rb/E2F complex and allowing E2F to promote cell-cycle progression to the S phase [16
]. The next step in adipogenesis is the re-entry of growth-arrested preadipocytes into the cell cycle and the completion of several rounds of MCE. Several groups have reported that MCE is necessary for subsequent preadipocyte differentiation [19
Adipocyte differentiation is a complex process that is mainly controlled by two families of transcription factors, CCAAT enhancer binding proteins (C/EBPs) and peroxisome proliferator-activated receptors (PPARs) [24
]. Differentiation of preadipocytes is characterized by marked changes in the pattern of gene expression that are caused by the sequential induction of these transcription factors. Preadipocytes exposed to differentiation inducers show an early and transient increase in expression of C/EBPβ and C/EBPδ [26
] which, in turn, contributes to cell proliferation and to the subsequent increase in expression of C/EBPα and PPARγ [28
]. These last two proteins are thought to act synergistically in the transcriptional activation of a variety of adipocyte-specific genes, with each activating the expression of the other [30
The other regulatory pathway of adipogenesis is the Wnt signaling pathway, which maintains preadipocytes in an undifferentiated state by inhibiting expression of the adipogenic transcription factors C/EBPα and PPARγ [32
]. In 3T3-L1 adipocytes, curcumin is reported to activate phosphorylation of AMP-activated protein kinase (AMPK), thus downregulating the PPARγ expression, and to inhibit adipogenesis [15
]. It also inhibits adipogenesis in 3T3-L1 adipocytes by restoring nuclear translocation of the Wnt signaling component β-catenin to a similar level compared to that in undifferentiated preadipocytes [34
As aforementioned, the nutrient constituents of curcumin make it a potential candidate for the therapy of obesity. However, this possibility and underlying mechanisms remain to be proven largely. Hence, this study aims to describe the role of curcumin in the regulation of adipocyte generation and sought to elucidate the molecular mechanisms of the actions responsible.
2. Methods and Materials
2.1. Experimental Design
To explore dose and time effects of curcumin on the viability of 3T3-L1 preadipocytes, the cells were pretreated for 1 h with different concentration of curcumin (0, 10, 15, 30, 50 μM), then incubated with differentiation medium (see below) in the continued presence of the same concentration of curcumin for different times (0, 24, 48, 72 h), when cell viability was determined using the MTT assay. To further clarify the effect of curcumin on apoptosis, 3T3-L1 preadipocytes were pretreated for 1 h with 30 μM curcumin, then incubated for 24 h with differentiation medium in the continued presence of 30 μM curcumin, then apoptosis was evaluated using the TUNNEL assay. To gain an insight into the apoptosis signaling pathway involved, expression of caspase proteins was examined by immunoblotting.
To explore the effect of curcumin on adipocyte differentiation, 3T3-L1 preadipocytes were pretreated for 1 h with different concentrations of curcumin (0, 5, 10, 15, 20 μM), then differentiation was induced by incubation for 10 days in the continued presence of the same concentration of curcumin. During the differentiation processes, MCE was measured by flow cytometry, phosphorylation or expression of adipogenic proteins (pRb, cyclin D1, C/EBPβ, p27, PPARγ, C/EBPα, and β-catenin) was measured by immunoblotting, and the efficiency of adipocyte differentiation was determined by measuring intracellular triglyceride (TG) content and by Oil red O staining.
To test whether the PPARγ ligand rosiglitazone reversed the inhibitory effect of curcumin on differentiation, 3T3-L1 preadipocytes were incubated for 1 h with or without 0.5 µM rosiglitazone, then were incubated with or without 15 μM curcumin in the continued presence or absence of rosiglitazone for 3 days in differentiation medium, 3 days in complete medium containing 1.7 μM insulin and 3 days in complete medium. During the differentiation processes, MCE and the efficiency of adipocyte differentiation were measured.
2.2. Cell Culture
3T3-L1 preadipocytes (American Type Culture Collection, Rockville, MD) were seeded onto 60-mm dishes or 12-well plates (Falcon®, Becton Dickinson, NJ, USA) and grown and maintained in complete medium [Dulbecco’s modified Eagle’s (DME) high glucose medium containing 100 units/mL of penicillin and 100 μg/mL of streptomycin (all from Gibco BRL, Gaithersburg, MD, USA), and 10% fetal bovine serum (FBS) (Biological Industries, Kibbutz Beit Ha’Emek, Israel)] in 10% CO2. The cells were grown to 3 days post-confluency, then induced to differentiate by incubation for 3 days in differentiation medium, i.e., complete medium containing 0.5 mM IBMX, 0.5 μM dexamethasone, and 1.7 μM insulin (all from Sigma, St. Louis, MO, USA), 3 days in complete medium containing 1.7 μM insulin and 3 days in complete medium. The medium was then changed every 3 days until the cells were fully differentiated. Typically, by day 10, >95% of the preadipocytes had differentiated into adipocytes as determined by staining for lipid accumulation using Oil Red O. This study protocol was used in all experiments.
2.3. Mitotic Clonal Expansion Assayed by Cell Counting or by the MTT Assay
Using direct cell counting, cell numbers were quantified after trypsinization using a hemocytometer. Using the MTT assay, cells were seeded onto 96-well plates at approximately 1 × 103 cells/well in 100 μL of DMEM containing 0.5% FBS. After incubation with curcumin, 15 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; USB, Amersham Life sciences, Cleveland, OH, USA) was added (final concentration 0.5 mg/mL) for 4 h, then 100 μL of DMSO was added to dissolve the formazan crystals formed and the optical density was measured on an ELISA plate reader using test and reference wavelengths of 570 and 630 nm, respectively.
2.4. Propidium Iodide Staining and Flow Cytometry Analysis
Treated cells were trypsinized, washed with phosphate-buffered saline (PBS), and fixed overnight in 75% ethanol. The cells were washed twice with PBS and the cell pellet resuspended in cold PBS, then the cells were incubated at room temperature for 5–10 minutes with PBS containing 25 μg/mL of propidium iodide (PI), 0.5 mg/mL of RNase A, and 0.1% Triton X-100. The fluorescence of the PI-stained cells was measured at 570 nm using a Cytomics FC 500 flow cytometer (Beckman Coulter Inc., Fullerton, California, USA) and the cell cycle distribution was analyzed using Cytomics FC 500 CXP software.
2.5. TUNEL Assay
DNA cleavage was verified by enzymatic end-labeling of DNA strand breaks using in situ cell death detection kits (Roche Applied Science, Indianapolis, IN) according to the manufacturer’s instructions. TUNEL-positive cells were evaluated on a Cytomics FC 500 flow cytometer (Beckman Coulter Inc., Fullerton, California, USA) and the data were analyzed using Cytomics FC 500 CXP software.
Whole cell lysates were prepared by sonication for 10 sec in an ice-bath in lysis buffer (1% Triton X-100, 50 mM KCl, 25 mM HEPES, pH 7.8, 10 μg/mL of leupeptin, 20 μg/mL of aprotinin, 125 μM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate). Samples (30 μg of total protein) in 50 μL of reducing sample buffer were boiled for 5 min and resolved on 12.5% SDS polyacrylamide gels for 90 min at 160 volts, then the proteins were transferred onto a polyvinylidene difluoride membrane for 120 min at 60 volts. The membrane was pre-blotted for 1 h at room temperature in blocking buffer (5% skimmed milk in PBS), then incubated for 24 h at 4 °C with primary antibodies against caspase 8, 9, or 3 (all from Cell Signaling Technologies, Danvers, MA, USA), PPARγ, C/EBPα, or β-catenin (all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), pRb, cyclin D1, C/EBPβ, p27, or β-actin (all from Sigma-Aldrich Corp., St. Louis, MO, USA) in blocking buffer, then for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody (Sigma-Aldrich Corp., St. Louis, MO, USA) in blocking buffer, followed by detection of bound antibody using chemiluminescence reagent (Amersham Biosciences, GE Healthcare, Bucks, UK). To detect multiple signals on a single membrane, the membrane was treated with stripping buffer (59 mM Tris-HCl, 2% SDS, 0.75% 2-mercaptoethanol) for 30 min at 50 °C prior to re-blotting with a different antibody.
2.7. Triglyceride Measurement
3T3-L1 adipocytes were homogenized by sonication and triglycerides were measured using commercial kits (DiaSys Diagnostic Systems GmbH & Co. KG, Holzheim, Germany).
2.8. Oil Red O Staining
To examine the lipid accumulation, cells cultured in 12-well plates were fixed with formalin and stained with Oil Red O. To quantify Oil Red O staining, the stained cells were washed with distilled water, and, after removal of the water, 1 mL of isopropanol was added for 10 min and the optical density was measured in a plate reader at 510 nm.
2.9. Statistical Analysis
Experiments were repeated four times. The results are expressed as the mean ± SD. Statistical significance was assessed by one way analysis of variance or Student’s t-test, a value of P < 0.05 being considered statistically significant.
The main finding of this study is that curcumin induced preadipocyte apoptosis in a time- and dose-dependent manner and that this cytotoxic effect involved the activation of both the intrinsic and extrinsic apoptotic pathways. Several studies have demonstrated that curcumin induces apoptosis in different types of cancer cell [35
]. These studies also demonstrated that curcumin induces cancer cell apoptosis through oxidative stress-, ER stress-, caspase cascade-, and mitochondria-dependent pathways. Our data are compatible with these studies. Besides, a recent study demonstrated that curcumin could induce apoptosis in SW872 human adipocytes [41
]. Ferguson et al. observed an increasing apoptotic signaling at concentrations greater than 30 μM [42
]. Our data show that high dose curcumin induces apoptosis in 3T3-L1 preadipocytes (Figure 1
and Figure 2
). In addition, we found that curcumin activates caspase-8 in 3T3-L1 preadipocytes. Lee et al. and Wang et al. found that curcumin could promote Fas and Fas ligand expression in cancer cells [36
]. Hence, it is possible that Fas/Fas ligand pathway may play a mechanistic role in curcumin-induced preadipocyte apoptosis. Many studies have demonstrated that curcumin suppresses adipocyte differentiation [13
], but its effect on the viability of preadipocytes was not investigated in these studies. An important finding of this work was that curcumin-induced preadipocyte apoptosis could contribute to the inhibitory effect of curcumin on adipogenesis.
Another finding of the present study was that low dose curcumin delayed MCE during the early stage of adipocyte differentiation (Figure 4
). The possible mechanism might involve the curcumin-induced downregulation of the early-response cell-cycle regulators phosphorylated Rb (pRb), cyclin D1, and p27Kip1
A,C). Such curcumin-induced interference with cell-cycle regulator levels might cause the delayed S phase entry and subsequent G2/M phase transition (Table 2
and Figure 1
B) and lead to delayed MCE. Studies have demonstrated that MCE is necessary for adipocyte differentiation [19
] and that the critical event for MCE is the transition from G1 phase to S phase [44
]. Thus, delayed S phase entry may cause inhibition of MCE and lead to suppression of adipogenesis in 3T3-L1 adipocytes. Our findings are also in agreement with those of several studies which showed that delayed cell cycle entry of 3T3-L1 preadipocytes during the early stage of differentiation decreases MCE and reduces the differentiation efficiency of adipocytes [45
]. Furthermore, we examined whether curcumin-inhibited adipogenesis was blocked by the addition of the PPARγ agonist rosiglitazone, which has been reported to directly bind and activate PPARγ and stimulate adipocyte differentiation [48
]. Rosiglitazone partially blocked curcumin-suppressed lipid accumulation in 3T3-L1 adipocytes (Figure 7
A), but had no effect on curcumin-induced delayed progression of MCE (Figure 7
B). These results provide indirect support for the idea that impairment of MCE might lead to the inhibition of adipocyte differentiation.
Cell-cycle regulators, such as Rb, cyclin D1, and p27Kip1
, play important roles in the G1 to S phase transition. In quiescent cells, there are low levels of cyclin D1 and high levels of p27Kip1
, which associates with the cyclin E-Cdk2 complex and suppresses Cdk2 enzyme activity. Mitogenic stimulation (early G1 phase) induces the synthesis of cyclin D1, resulting in increased formation of the cyclin D/Cdk4 complex, then p27Kip1
dissociates from the cyclin E-Cdk2 complex and binds to the cyclin D/Cdk4 complex and the Cdk2 activity is enhanced. Activated Cdk2 and Cdk4 phosphorylate Rb, pRb dissociates from E2F1, E2F1-dependent genes are expressed, and the cell cycle starts. Activated cyclin E-Cdk2 also phosphorylates p27Kip1
, triggering its ubiquitination and degradation [50
]. In the present study, the pattern of Rb phosphorylation and cyclin D1 and p27Kip1
expression in the control cells matched those in control differentiating cells during the early stage of adipocyte differentiation reported previously [51
]. However, treatment with 15 μM curcumin significantly decreased Rb phosphorylation and cyclin D1 expression and increased the P27Kip1
expression in the differentiating cells. Tian et al. also reported that treatment of curcumin could induce Rb mRNA upregulation in 3T3-L1 adipocytes [52
]. These curcumin-induced changes in the expression profiles of cell cycle regulators might cause delayed G1/S phase transition and lead to the observed reduced MCE and suppressed adipocyte differentiation. Kim et al. [43
] reported that curcumin modulates MCE by downregulating cyclin A and Cdk2 levels. During the G1/S phase transition, mitogenic stimulation induces cyclin D1 synthesis, followed sequentially by Rb phosphorylation and Cdk2 activation [50
]. In our study, the curcumin-induced decrease in Rb phosphorylation and cyclin D1 expression and increase in P27Kip1
expression occurred earlier (G1 phase) than in Kim’s study (S phase), but both studies suggest that curcumin interferes with MCE, then inhibits adipogenesis.
Phosphorylation of Rb is a major step in E2F1 activation, as Rb phosphorylation leads to Rb dissociation from transcription factor E2F1, triggering cell cycle progression [50
]. Similar events occur during the early stage of adipogenesis [54
]. A previous study demonstrated that E2F1 can regulate 3T3-L1 adipogenesis by binding directly to the PPARγ promoter and increasing expression of PPARγ, the master regulator of adipocyte differentiation [51
]. In addition, a crosstalk between PPARγ and Rb signaling might operate during adipocyte differentiation, as a study has shown that Rb recruits histone deacetylase 3 (HDAC3) to PPARγ target genes and that disruption of the PPARγ-Rb-HDAC3 complex by Rb phosphorylation or inhibition of HDAC3 activity results in activation of PPARγ, translating as an increase in adipogenesis [55
]. In addition, pRb has been shown to interact with the members of the C/EBP family, including C/EBPα and C/EBPβ [16
]. These findings indicate that pRb plays a positive and direct role in the preadipocyte proliferation and terminal adipocyte differentiation.
In addition to the curcumin-induced downregulation of PPARγ and C/EBPα
, we also found that differentiation medium-induced downregulation of β-catenin was prevented by low dose curcumin treatment (Figure 6
A,D). Previous studies has demonstrated that upregulation and activation of the Wnt/β-catenin pathway in 3T3-L1 preadipocytes inhibits adipocyte differentiation [32
]. In the present study, curcumin abolished the differentiation medium-induced downregulation or suppression of Wnt/β-catenin signaling and this may contribute to the curcumin-induced inhibition on adipocyte differentiation. A very similar finding was reported by Ahn et al. [34
], who demonstrated that curcumin stimulates the expression of Wnt/β-catenin signaling components and targets in differentiating adipocytes. With the exception of the β-catenin expression results, our study and that of Ahn et al. both show that curcumin might suppress adipogenesis through modulation of Wnt/β-catenin signaling.
Results of our present study are compatible with the findings of several studies, including curcumin inhibits adipocyte differentiation through modulation of MCE [43
] and activation of Wnt/β-catenin signaling [34
]. In conclusion, the present study demonstrates that curcumin has dual effects on the regulation of adipogenesis (Figure 8
). High dose curcumin induces preadipocyte apoptosis in a time- and dose-dependent manner through caspase 3- 8-, and 9-dependent pathways. In addition, low dose curcumin inhibits adipocyte differentiation by altering the expression of cell cycle regulators, reducing MCE, downregulating expression of PPARγ and C/EBPα, preventing differentiation medium-induced β-catenin downregulation, and decreasing lipid accumulation. These findings suggest that curcumin supplementation could be an effective strategy for treating or preventing development of obesity by a curcumin-induced reduction in the number of preadipocytes and the fat mass of adipocytes.