Apigenin isolated from Daphne genkwa Siebold et Zucc. inhibits 3T3-L1 preadipocyte differentiation through a modulation of mitotic clonal expansion
Introduction
Obesity is a public health threat worldwide because it is a major risk factor for type 2 diabetes mellitus, hypertension, atherosclerosis, cancer, and cardiovascular disease (Calle et al., 2003, Kannel et al., 1991). There is considerable research interest in the discovery of compounds with anti-obesity activity, particularly those that act through appetite suppression, inhibition of nutrient absorption, increased energy expenditure, or modulation of fat storage (Bray and Tartaglia, 2000, Wang et al., 2004). Currently approved drugs for the long-term treatment of obesity include sibutramine, which inhibits food intake, and orlistat, which blocks fat digestion, but both have undesirable side effects. Accordingly, dietary bioactives derived from natural products are attractive alternatives to synthetic anti-obesity drugs. The main criteria for studying underlying mechanisms of anti-obesity phytochemicals are inhibition of preadipocyte proliferation and differentiation, inhibition of lipogenesis, activation of lipolysis, and increased fat oxidation (Rayalam et al., 2008). Therefore, the identification of natural products, together with an understanding of their underlying mechanisms of action, may help to prevent the initiation and progression of obesity and its associated diseases.
Adipogenesis involves two major events: preadipocyte proliferation, and adipocyte differentiation. The transition between both processes is a tightly regulated process in which cell cycle regulators and differentiating factors interact, creating a cascade of events leading to preadipocyte commitment to the adipocyte phenotype (Fajas, 2003). Since the development of the murine adipose 3T3 cell culture system, 3T3-L1 and 3T3-F442A cells have become a popular in vitro model for studying adipocyte differentiation (Green and Kehinde, 1975). In the presence of a standard adipogenic cocktail comprising 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, and insulin, growth-arrested 3T3-L1 preadipocytes synchronously re-enter the cell cycle, undergo two rounds of mitosis (known as the mitotic clonal expansion (MCE)), and then exit the cell cycle and commence terminal differentiation (characterized by morphological changes, lipid accumulation, and the expression of almost all genes characteristic of fat cells) (Tang et al., 2003a, Tang et al., 2003b, Tang and Lane, 1999). Several members of the CCAAT/enhancer binding proteins (C/EBP family), as well as peroxisome proliferator-activated receptor γ (PPARγ), participate in a transcriptional cascade during adipogenesis (Rosen et al., 2002, Rosen and Macdougald, 2006). Although C/EBPβ is expressed immediately on induction, it requires a long lag period (~ 14 h) for localization to centromeres and DNA-binding activity, which are critical for the expression of two principal adipogenic factors, C/EBPα and PPARγ (Tang et al., 2003a, Tang et al., 2003b, Macdougald and Lane, 1995, Christy et al., 1991).
Recently, the clinical importance of herbal drugs has received considerable attention, and flavonoids, which are often found in herbal drugs and foods, are known to possess many useful biological properties. The dried flowers of Daphne genkwa Siebold et Zuccarini (Thymelaeaceae) (“Genkwa Flos”), a Chinese herbal medicine distributed primarily in mainland China and Korea, are traditionally used for their abortifacient, anticancer, antitussive, diuretic, and anti-inflammatory effects (Zhou, 1991, Hong et al., 2011, Bae et al., 2012). The present study examined several compounds isolated from the flavonoid-rich fraction of D. genkwa Siebold et Zuccarini crude extracts (GFFs) for their ability to inhibit the differentiation of 3T3-L1 cells into adipocytes. Only apigenin potently inhibited 3T3-L1 differentiation. Apigenin (5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) is a naturally occurring plant flavone that is abundant in common fruits and vegetables; it is also a bioactive flavonoid that possesses anti-inflammatory, antioxidant, and anticancer properties (Nielsen et al., 1999, Yang et al., 2001, Shukla and Gupta, 2010, Patel et al., 2007). Although obesity is associated with increased oxidative stress and inflammation in adipose tissue, the mechanisms underlying the beneficial role of apigenin during adipose tissue development are still under investigation. Previous studies show that apigenin impairs 3T3-L1 preadipocyte differentiation (Bandyopadhyay et al., 2006, Phrakonkham et al., 2008). Furthermore, apigenin inhibits food intake in C57BL/6J mice (Myoung et al., 2010). Recently, Ono and Fujimori demonstrated that apigenin mediates anti-adipogenic effects by activating AMPK in 3T3-L1 cells (Ono and Fujimori, 2011). However, the molecular mechanisms by which apigenin acts during adipogenesis are not fully understood.
Here, we examined the role of apigenin and attempted to identify the mechanisms underlying its effects on adipogenic differentiation using 3T3-L1 cells. We found that GFF inhibited the proliferation and differentiation of 3T3-L1 cells. Apigenin, the active ingredient in GFF, suppressed preadipocyte growth and differentiation by inhibiting MCE; specifically, it induced cell cycle arrest at the G0/G1 phase during the early stages of adipocyte differentiation. The mechanisms by which apigenin inhibited MCE include down- and up-regulations of positive (such as cyclin D1 and cyclin-dependent kinase (CDK) 4) and negative cell cycle regulators (p27Kip1), respectively. Apigenin also down-regulated the expression of the adipogenic transcriptional factors, C/EBPα, C/EBPβ, and PPARγ. In addition, apigenin inhibited C/EBPβ activity in differentiating 3T3-L1 cells by up-regulating the expression of two different sets of C/EBP inhibitors: the phospho-liver-enriched inhibitory protein (LIP) isoform of C/EBPβ and C/EBP homologous protein (CHOP)-10.
Section snippets
Materials
Dulbecco's-modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Hyclone (South Logan, UT, USA) and newborn calf serum (NBCS) was purchased from Gibco (Grand Island, NY, USA). Insulin, dexamethasone, IBMX, troglitazone, propidium iodide (PI), and Oil Red O were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Plant material
Dried flower buds of D. genkwa were purchased from the Kyungdong oriental herbal market, Seoul, Korea, and identified by one of the authors (C.Y. Kim). The voucher
GFF and flavonoids isolated from GFF inhibit adipogenic cell differentiation
To test the anti-adipogenic potential of GFF, 3T3-L1 cells were stimulated with a standard adipogenic cocktail (dexamethasone, IBMX, and insulin) together with a PPARγ agonist and GFF (25, 50, 75 or 100 μg/mL). The effects on adipogenesis in 3T3-L1 cells were measured using Oil Red O staining, and cellular lipid accumulation was determined after differentiation. As shown in Fig. 1A, GFF decreased lipid accumulation in a dose-dependent manner at concentrations of 25, 50 and 75 μg/mL, with no
Discussion
Obesity develops when energy intake chronically exceeds total energy expenditure. Excessive caloric intake relative to expenditure produces a metabolic state that promotes hyperplasia (increase in number) and hypertrophy (increase in size) of adipocytes (Shepherd et al., 1993). The rise in adipocyte number involves the conversion of mesenchymal stem cells to preadipocytes, which then differentiate into adipocytes. All anti-obesity medications currently approved by the FDA act to repress energy
Conclusions
In summary, we showed that GFF inhibits the adipogenesis of 3T3-L1 cells, and that apigenin is the active ingredient responsible for these effects. Furthermore, apigenin inhibits adipogenesis by targeting the early biochemical and cellular events that occur during adipogenesis, including MCE. Apigenin down-regulates the expression of the early adipogenic transcriptional factors, C/EBPβ, PPARγ and C/EBPα, and up-regulates the expression of multiple C/EBPβ inhibitors, including the phosphorylated
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgments
This work was supported by an intramural grant (2Z03850) from the Korea Institute of Science and Technology, Gangneung Institute.
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