A clerodane diterpene inhibit adipogenesis by cell cycle arrest and ameliorate obesity in C57BL/6 mice
Introduction
Obesity is one of the most prevalent forms of disorder widespread throughout the globe. It is the key contributing factor for various pathological states such as type 2 diabetes, hyperlipidemia, hypertension and arteriosclerosis and metabolic syndrome (Kopelman, 2000). The widely used measure of obesity is body mass index (BMI) that has to be greater than 30 kg/m2 (Calle et al., 2003). Both adipocyte hyperplasia and hypertrophy are determinant factors for obesity caused by imbalance of energy intake and expenditure (Spiegelman and Flier, 1996).
For in vitro experiments, 3T3-L1 pre-adipocyte has served as an excellent model system to study different aspects of adipogenesis. In this model, upon hormonal induction, C/EBPβ is rapidly expressed, and growth arrested cells synchronously re-enter the cell cycle traversing the G1–S checkpoints to initiate Mitotic clonal expansion (Tang et al., 2003a). C/EBPβ is thought to mediate the expression of PPARγ and C/EBPα (Hishida et al, 2009, Yeh et al, 1995). Both the proteins are pleiotropic transcriptional activators that coordinate to induce expression of adipocyte genes to drive adipogenesis (White and Stephens, 2010).
Prominent among the factors that modulate adipogenesis in the early stage, is mTOR pathway that is required for differentiation of pre-adipocyte and primary cells (Xiang et al., 2011). Rapamycin, a known mTORC1 inhibitor, inhibits mitotic clonal expansion, decreases expression of PPARγ and C/EBPα and inhibits adipogenesis (Gagnon et al., 2001). Knockdown of Raptor leads to reduction in mTOR as well as adipogenesis (Polak et al., 2008). Increased mTOR activity play a permissive role in adipogenesis by augmenting upregulation of PPARγ and C/EBPα (Zhang et al., 2009). mTOR, also positively regulate mitochondrial oxygen consumption in mammalian cells (Schieke et al., 2006). Mitochondrial oxygen consumption and overall ROS level increases during early events of mesenchymal stem cell adipogenesis. N-acetyl cysteine (NAC) and reactive oxygen species (ROS) reducing agent supplementation decrease adipogenesis (Tormos et al., 2011). These studies underscore the importance of mTOR and ROS in early adipogenesis.
There are currently few FDA approved anti-obesity drugs in the market including Sibutramine (appetite suppressor), Orlistat (gastrointestinal lipase inhibitor), and fibrates (PPARα agonists) (Padwal and Majumdar, 2007). Despite these available options, worldwide incidences of obesity are increasing at the alarming rate. Natural products provide a rich source of chemical diversity that can be used to design and develop new drug leads. Some of the natural products including epigallocatechin gallate (EGCG), berberine and curcumin, are known to suppress adipogenesis and proved useful in obesity management in animal models (Ejaz et al, 2009, Hu, Davies, 2010, Lee et al, 2013, Meydani, Hasan, 2010).
P. longifolia var. pendula Linn. belongs to the family Annonaceae and has shown various pharmacological properties (Jothy et al, 2012, Malairajan et al, 2008, Mandal et al, 2012, Manjula et al, 2010). Phytochemical studies on the ethanolic extract of the leaves of P. longifolia have led to the characterization of various clerodane diterpenes. Recent studies from our group have demonstrated that diterpene 16α-hydroxycleroda-3, 13 (14)Z-dien-15, 16-olide (compound 1) isolated from Polyalthia longifolia var. pendula leaves is a major hypolipidemic constituent (Sashidhara et al., 2011). The pharmacokinetic study demonstrated that compound 1 shows rapid gastro-intestinal absorption and was in the systemic circulation for 48 h following a single dose oral administration (Bhatta et al., 2012). In vitro assays demonstrated significant HMG-CoA reductase inhibition and molecular docking studies proved this clerodane diterpene to be comparable with lovastatin. Recently, we have observed co-existence of anti-dyslipidemic/hypolipidemic activities with anti-adipogenic activity for various flavanoids although both activities are distinct (Varshney et al., 2014). Further to this, some of the statin classes of compounds were also reported for anti-adipogenic activity (Nakata et al., 2006). These observations prompted us to undertake studies on anti-adipogenic effect of compound 1 (the diterpene class) and comparative assessment with another statin class of compounds (Polyketide class).
Our results show, diterpene compound 1 although belonging to a distinct chemical class, possess anti-adipogenic activity similar to statin classes of compounds. The activity is mediated through modulations of early signaling, resulting in Mitotic clonal expansion arrest, followed by suppression of adipogenic regulators. Anti-adipogenic activity was also confirmed using other in vitro models of adipogenesis including a mouse stromal stem cell line C3H10T1/2, and hMSCs. Compound 1 also demonstrated in vivo anti-obesity activity similar to Orlistat, the FDA approved anti-obesity drug.
Section snippets
Plant material extraction and isolation
Polyalthia longifolia var. pendula leaves were collected from Lucknow in 2005. The identity of the plant was confirmed and a voucher specimen (No. 6381) has been deposited in the herbarium of the Botany Division, CSIR-Central Drug Research Institute (CSIR-CDRI), Lucknow, India. Compound 1 isolation (Sashidhara et al., 2011), spectrophotometric and HPLC purity analysis were performed as briefly mentioned in supporting information.
Cell culture
3T3-L1 mouse embryo fibroblasts were obtained from American Type
Results
Recently we have shown observed inter-dependence of anti-dyslipidemic and anti-adipogenic activities in many of the flavonoid class of compounds (Varshney et al., 2014). Some of the statin classes of anti-dyslipidemic compounds also exhibit anti-adipogenic activities. Taking clues from these studies, we screened in house natural product library showing anti-dyslipidemic activities and identified compound 1 as potential anti-adipogenic hit molecule.
Discussion
In our recent work we reported observed inter-relationship between compounds reported for in vitro anti-adipogenic activity also demonstrating anti-dyslipidemic activity in vivo (ref). Although our earlier reported study covered mainly flavonoid class of compounds (Varshney et al., 2014), interestingly statin class of compounds were also reported for exhibiting anti-adipogenic activity. Taking clues, we reverse screened our compound library molecules demonstrating anti-dyslipidemic activity for
Conclusion
In summary, our studies provide the first evidence that Compound 1 inhibits adipogenesis in 3T3-L1 cells by blocking early MDI induced signaling. Furthermore Compound 1 like Orlistat ameliorates obesity, weight gain, impaired glucose tolerance, inflammatory markers and lipid parameters when given in-vivo (Fig. 6). Taken together, earlier reported anti-dyslipidemic alongwith anti-adipogenic and anti-obesity activities observed herewith, Compound 1 is a good translational lead candidate for
Acknowledgements
Research work is supported by CSIR-CDRI Network project: “Towards holistic understanding of complex diseases: Unraveling the threads of complex disease (THUNDER, Grant: BSC0102)”. Authors acknowledge experimental support from Flow Cytometry facility of SAIF-CDRI and Mr. P. K. Singh of CSIR-IITR. This manuscript bears CSIR-CDRI communication number: 8799.
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