A withanolide coagulin-L inhibits adipogenesis ...

1 downloads 0 Views 2MB Size Report
A withanolide coagulin-L inhibits adipogenesis modulating. Wnt/-catenin pathway and cell cycle in mitotic clonal expansion. Muheeb Bega, Parul Chauhanb, ...
Phytomedicine 21 (2014) 406–414

Contents lists available at ScienceDirect

Phytomedicine journal homepage: www.elsevier.de/phymed

A withanolide coagulin-L inhibits adipogenesis modulating Wnt/␤-catenin pathway and cell cycle in mitotic clonal expansion Muheeb Beg a , Parul Chauhan b , Salil Varshney a , Kripa Shankar a , Sujith Rajan a,c , Deepika Saini b , M.N. Srivastava d , Prem P. Yadav b,∗ , Anil Nilkanth Gaikwad a,c,∗ a

Division of Pharmacology, CSIR-Central Drug Research Institute, Lucknow 226031, India Division of Medicinal and Process Chemistry, CSIR-Central Drug Research Institute, Lucknow 226031, India c Academy of Scientific and Innovative Research, CSIR-CDRI, India d Division of Botany, CSIR-Central Drug Research Institute, Lucknow 226031, India b

a r t i c l e

i n f o

Article history: Received 3 July 2013 Received in revised form 3 September 2013 Accepted 11 October 2013 Keywords: Coagulin-L Withania coagulans Mitotic clonal expansion Obesity 3T3-L1 Adipocytes Adipogenesis

a b s t r a c t Obesity is a result of adipocyte hypertrophy followed by hyperplasia. It is a risk factor for several metabolic disorders such as dyslipidemia, type-2 diabetes, hypertension, and cardiovascular diseases. Coagulanolides, particularly coagulin-L isolated from W. coagulan has earlier been reported for anti-hyperglycemic activity. In this study, we investigated the effect of coagulin-L on in vitro models of adipocyte differentiation including 3T3-L1 pre-adipocyte, mouse stromal mesenchymal C3H10T1/2 cells and bone marrow derived human mesenchymal stem cells (hMSCs). Our results showed that, coagulin-L reduces the expressions of peroxisome proliferator-activated receptor ␥ (PPAR␥) and CCAAT/enhancer-binding protein ␣ (C/EBP␣), the major transcription factors orchestrating adipocyte differentiation. Detailed analysis further proved that early exposure of coagulin-L is sufficient to cause significant inhibition during adipogenesis. Coagulin-L inhibited mitotic clonal expansion (MCE) by delayed entry in G1 to S phase transition and S-phase arrest. This MCE blockade was caused apparently by decreased phosphorylation of C/EBP␤, modulation in expression of cell cycle regulatory proteins, and upregulation of Wnt/␤-catenin pathway, the early stage regulatory proteins of adipogenic induction. Taken together all evidences, a known anti-hyperglycemic agent coagulin-L has shown potential to inhibit adipogenesis significantly, which can be therapeutically exploited for treatment of obesity and metabolic syndrome. © 2013 Elsevier GmbH. All rights reserved.

Introduction Obesity and associated metabolic syndrome has become a worldwide health problem, with overweight population exceeding 1.7 billion (Muoio and Newgard, 2006; Stein and Colditz, 2004). Obesity initiates with adipocyte hyperplasia and hypertrophy, and is mimicked ex vivo by the hormone induced differentiation of 3T3-L1 pre-adipocytes. After hormonal induction, CCAAT/enhancer binding protein-␤ (C/EBP␤) is rapidly expressed and growth-arrested preadipocytes synchronously re-enter the cell cycle traversing G1–S checkpoints, and initiate MCE. This sets in increased expression of peroxisome proliferator-activated receptor ␥ (PPAR␥) and C/EBP␣, that co-ordinately induce expression of

∗ Corresponding authors at: BS 10/1, Sector 10, CSIR-Central Drug Research Institute, Jankipuram Extension, Lucknow 226031, Uttar Pradesh, India. Tel.: +91 522 2771940x4876; fax: +91 522 2771941. E-mail addresses: pp [email protected] (P.P. Yadav), anil [email protected] (A.N. Gaikwad). 0944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2013.10.009

genes associated with adipogenesis (Tang et al., 2003). Resveratrol and its metabolite piceatannol inhibit adipogenesis by targeting MCE and insulin signaling (Kwon et al., 2012a,b). Thus, MCE is a necessary step for the terminal adipocyte differentiation. Pharmacological and genetic studies provide evidence that mTOR plays positive role in adipogenesis (El-Chaar et al., 2004; Kim and Chen, 2004). Earlier studies suggested that mTOR control adipogenesis/lipogenesis by PPAR␥ and C/EBP␣ (Blanchard et al., 2012; Zhang et al., 2009; Jung et al., 2013; Takahashi et al., 2012). The other important pathway that modulates adipogenesis negatively, is Wnt/␤-catenin (Kawai et al., 2007). Curcumin, EGCG and (+)-Episesamin modulate Wnt/␤-catenin pathway and inhibit adipogenesis (Ahn et al., 2010; Freise et al., 2013; Lee et al., 2013). Natural products are rich source of chemical entities that control obesity, either by inhibiting adipogenesis, increasing lipolysis, inducing apoptosis of adipocytes or combinations thereof (Rayalam et al., 2008). Withania coagulans Dunal (family: Solanaceae), has been highlighted in Ayurveda for treatment of type 2 diabetes. W. coagulans has been reported for its hepatoprotective and hypolipidemic activities (Hemalatha et al., 2006). Previous chemical

M. Beg et al. / Phytomedicine 21 (2014) 406–414

investigation of this plant resulted in the isolation and identification of several withanolides (Maurya et al., 2008). Amongst isolated withanolides, the major constituent coagulin-L has shown most significant hypoglycemic activity in streptozotocin induced diabetic rats and db/db mice (Maurya et al., 2008). Hyperglycemia promotes lipid accumulation to maintain homeostasis (Chuang et al., 2007). Atorvastatin, Simvastatin, Berberine and Silibinin, that possess hypo-lipidemic and hepato-protective activities, have also been shown to inhibit lipid accumulation in adipocytes (Huang et al., 2006; Ka et al., 2009; Tomiyama et al., 1999). Withanolides isolated from W. coagulans has similarly reported biological activities, but none of its constituents have so far been studied for its effects on adipogenesis. We therefore, evaluated abundant and most active withanolide of Withania coagulans, i.e., coagulin-L for its effects on adipogenesis. Our results indicate that coagulin-L inhibit early stage differentiation by inhibiting expression of C/EBP␤, mTOR and associated protein phosphorylation. It reduces expression of G1-S phase specific cells cycle markers and block MCE. This, in turn represses PPAR␥ and C/EBP␣ and their down-stream target genes participating in adipogenesis. Our results demonstrate anti-adipogenic potential of coagulin-L mediated modulation of early signaling events during adipogenesis. Materials and methods Reagents Dulbecco’s modified Eagle’s medium (DMEM), and fetal bovine serum were purchased from GIBCO-BRL Life Technologies (NY, USA). Insulin, 3-isobutyl-1-methylxanthine, dexamethasone, and Oil Red-O (ORO) dye were purchased from Sigma Chemical (St Louis, MO). Antibodies against PPAR␥, C/EBP␣, C/EBP␤, GLUT4, pmTOR, p-C/EBP␤, p-P70S6K, p-P44/42, p-Akt, CDK6 and LRP were purchased from Cell Signaling Technology (Beverly, MA). CDK2, CDK4, p27, and ␤-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All the chemicals were of analytical grade.

407

Fig. 1. Structure of coagulin-L from Withania coagulans.

of 0.5 mM 3-isobutyl-1-methylxantine, 250 nM dexamethasone, and 5 ␮g/ml insulin in 10% fetal bovine serum for the first 48 h. The medium was then changed with DMEM containing 10% fetal bovine serum and 5 ␮g/ml insulin, and the cells were cultured for 2 days. The cells were then incubated in DMEM supplemented with 10% fetal bovine serum till cell achieve full adipocyte morphology. To study effect of compound on adipogenic differentiation, compound was added throughout differentiation at given concentration mentioned in figures. Atorvastatin was used as positive control. Similarly mouse mesenchymal stem cells C3H10T1/2 (ATCC Catalog number CCL-226) and bone marrow derive human mesenchymal stem cell (hMSC) (Stempuetics, Batch No. BMMSC1104) were cultured in DMEM containing 10% (v/v) fetal bovine serum and differentiated in MDI supplemented with 1 ␮M Rosiglitazone for initial 72 h. After that media replaced with culture media supplemented with 5 ␮g/ml insulin and 1 ␮M Rosiglitazone till the cell achieve full morphology of adipocyte.

Plant collection, extraction and isolation of coagulin-L

ORO staining

Withania coagulans Dunal (Family: Solanaceae) fruits were collected locally in November 2011 and identified at Botany Division of CSIR-Central Drug Research Institute, Lucknow. A voucher specimen (No. 6487) is kept in the herbarium for future reference. The dried fruits of W. coagulans (8 kg) were extracted by cold percolation from 95% ethanol. The combined alcoholic extract was concentrated under reduced pressure using rotavapor at 45 ◦ C to a brown gum ethanol extract (1.5 kg). Ethanol extract was dissolved in water and partitioned successively with ethyl acetate to afford ethyl acetate fraction (500 g) and water fraction (800 g). The aqueous fraction was subjected to repeated column chromatography over silica (60–120 mesh) and flash silica (230–400 mesh). Finally coagulin-L (2 g) was purified by using reverse phase (LiChroprep RP-18, 25–40 ␮m) flash column chromatography using a gradient of methanol:water (1.5:8.5–1:1) and characterized by comparison of spectroscopic data reported in literature Rahman et al. (1998). The chemical structure of the coagulin-L used in this study is shown in Fig. 1.

Fully differentiated adipocytes (with or without compound) were rinsed in phosphate buffered saline (PBS, pH 7.4). The adipocytes lipid globules were stained with ORO (0.36% in 60% isopropanol) for 20 min. Unstained ORO was removed by rinsing wells twice with PBS. After complete removal of PBS, 40× images were acquired on Nikon Ti microscope and the dye was extracted from cells using 100% isopropanol and measured absorbance at 492 nm.

Cell culture and adipogenesis induction 3T3-L1 (American Type Culture Collection (ATCC; Manassas, VA, Catalog number CL-173) preadipocytes were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal bovine serum. Post-confluent 3T3-L1 cells were induced to differentiate by the addition of differentiation media (MDI) composed

Cell proliferation assay To assess cell proliferation, 3 H-thymidine incorporation was measured after induction of MDI in confluent 3T3-L1 pre-adipocyte for 48 h. Briefly, cells were washed three time with chilled PBS. Cells were lysed in 200 ␮l of 2 N NaOH for over 2 h and samples were analyzed by a scintillation counter (LS6500, Beckman Coulter, USA). Western blotting Cells were lysed in ice-cold mammalian lysis buffer containing 0.5 M EDTA, protease inhibitor (Amresco) and phosphatase inhibitor Phos-STOP (Roche). Protein concentrations were measured by Bicin-choninic acid method (Sigma). Protein lysates were denatured by heating at 65 ◦ C for 10 min. in laemmli sample buffer supplemented with 10% ␤mercaptoethanol and protein was resolved by 8–12% SDS-PAGE and electro-transferred to nitrocellulose paper at 50 V for 2 h. The membranes were blocked for

408

M. Beg et al. / Phytomedicine 21 (2014) 406–414

Table 1 Primer sequences for real-time RT-PCR. Gene Name

Primer(s)-forward (F) reverse (R)

LPL

F 5 tttgtgaaatgccatgacaag3 R 5 cagatgctttcttctcttgtttgt3 F 5 gaaaacgagatggtgacaacg3 R 5 gccctttcataaactcttgtgg3 F 5 ttcctcagactgtaggcaaatct3 R 5 agcctcagtttacccactcct3 F 5 caacatgggacaccctgag3 R 5 gttgtggaagtgcaggttagg3 F 5 aagacaacggacaaatcacaa3 R 5 gggggtgatatgtttgaacttg3 F 5 aaacaacgcaacgtggaga3 R 5 gcggtcattgdcactggtc3 F 5 gagacatggggacacagtca3 R 5 Gggaatcagatgggtcctg3 F 5 cacccctatcccgtgaatc3 R 5 cagcagtagagagtaagagacacca3 F 5 tgttaccaactgggacgaca3 R 5 ggggtgttgaaggtctcaaa3

aP2 SREBP-1c FAS-n PPAR-␥CEBP-␣Wnt3aGATA2GAPDH-

1 h at room temperature in 5% skimmed milk (Sigma) in trisbuffered saline containing 0.05% Tween-20 (TBS-T). After washing with TBS-T, the membranes were incubated with target protein specific antibodies for overnight at 4 ◦ C, followed by incubation with appropriate HRP-conjugated secondary antibodies for 1 h. The target proteins were detected using Immobiline western Chemiluminescence detector (Millipore; Billerica, USA) on Image Quant LAS 4000. To validate equal loading in each lane, ␤-actin was used as internal loading control. Cell cycle analysis using flow cytometry 3T3-L1 pre-adipocyte cell grown to confluence in T25-flasks. Two day post-confluent cells were incubated in medium for differentiation induction with or without coagulin-L for 16 h and 24 h at 10 ␮M and 15 ␮M concentration. For cell cycle arrest studies resveratrol (50 ␮M) was used as positive control. The cells were harvested after 16 h and 24 h, washed in PBS and re-suspended in 1 ml PBS. Then cells were fixed in 70% ethanol for 2 h on ice. Pelleted cells were suspended in propidium iodide solution (500 ␮g/ml made in RNAase A containing buffer) for 30 min. at room temperature. At least 10,000 events were acquired per sample on flow cytometer (BD, FACS Calibur). Analysis was performed using Modfit software to determine the relative amount of cells in G1, S and G2/M phase. Real time PCR Total RNA was isolated from 3T3-L1 cells using TRIZOL reagent (Invitrogen, CA, USA). First strand cDNA synthesis performed using high capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) and subsequently used for quantitative real time PCR analysis on Light Cycler 480 (Roche Diagnostics) using SYBR Green master mix (Roche Diagnostics). Statistical analysis of the quantitative real time PCR obtained using the (2−Ct ) method, which calculates the relative changes in gene expression of the target normalized to an endogenous reference (GAPDH) and relative to a calibrator that serves as the control group. The list of gene specific primer pairs used in this experiment are listed in Table 1. Statistical analysis Data were expressed as mean ± SD and Student’s t-test was used for comparisons of measured parameters. p-Values 0.05) with lipid accumulation decreasing gradually with maximum inhibition at 0–6 days. Coagulin-L exposure during maturation stages 2–4, 2–6 and 4–6 days lead to 20–40% decreased accumulation of lipids when compared to control in 3T3-L1 cells (Fig. 4a and b). Similarly, gradual time dependent effects were also observed with C3H10T1/2 cell line (Fig. 4a and c). These results suggested that early time point exposure, i.e., 0–2 days, was required and sufficient to achieve significant suppression of lipid accumulation. Coagulin-L suppresses MDI induced early signaling As indicated above, the exposure of coagulin-L for initial 48 h was critical for the suppression of adipogenesis. C/EBP␤ gets expressed and undergoes phosphorylation during the early stage of adipogenesis. Addition of coagulin-L did not altered expression of C/EBP␤, but reduced phosphorylation of C/EBP␤, mTOR and P70S6K

M. Beg et al. / Phytomedicine 21 (2014) 406–414

409

Fig. 2. Coagulin-L inhibit in vitro adipogenesis in concentration dependent manner. (a) Microscopic images of oil-red O stained adipocytes those differentiated in presence of different concentrations of coagulin-L and standard anti-adipogenic compound atorvastatin. (b) Absorbance of extracted ORO quantified at 490 nm. (c and e) Coagulin-L mediated adipogenic inhibition in murine stem cell line C3H10T1/2 and hMSC derived adipocytes. (d and f) Extracted ORO absorbance from C3H10T1/2 and hMSC derived adipocytes respectively. Results expressed are mean ± SD of three exp. with statistical significance *p < 0.05, **p < 0.01 and ***p < 0.001.

410

M. Beg et al. / Phytomedicine 21 (2014) 406–414

Fig. 3. Molecular markers of adipogenesis inhibition. Effect of coagulin-L on the phosphorylation of mTOR (Ser2448), P70S6K (Thr389) and 4EBP (Thr37/46) and expression level of PPAR␥, C/EBP␣ and GLUT4 analyzed on days 2, 4 and 6 of adipogenic differentiation. ␤-actin used as loading control. Proteins run on same gel but on different position. (b) Relative mRNA abundance of GATA2 (day 2) and aP2, FAS, LPL, PPAR␥, SREBP-1c (day 4) performed by real-time PCR in untreated and coagulin-L treated differentiating adipocytes. Normalization performed by GAPDH. Values expressed are mean ± SD of two exp. in triplicate with statistical significance *p < 0.05, **p < 0.01 and ***p < 0.001.

at 16 h and 24 h in 3T3-L1 cells (Fig. 5c). Apart from this, addition of coagulin-L also inhibited MDI induced phosphorylation of ERK, mTOR and 4EBP during 15 min to 2 h (Fig. 5d). However, coagulin-L did not have any impact on MDI induced AKT phosphorylation in 3T3-L1 cells.

Coagulin-L induces S-phase arrest through P27 and CDKs Flow cytometry results showed that coagulin-L treated cells displayed delayed cell cycle progression at both 16 h and 24 h after MDI induction. At 16 h time point, coagulin-L showed decrease in S-phase and increase in G1-phase cells in concentration dependent manner. At 16 h 62.25% cells were in the S-phase, coagulin-L treatment decreased S-phase to 32.6% and 15.37% at 10 ␮M and 15 ␮M concentrations, respectively. G1-phase increased from 33.97% to 52.35% at 10 ␮M and 62.92% at 15 ␮M. At 24 h time point coagulinL showed significant increase in S-phase from 24.9% to 40.5% and 42.33% at 10 ␮M and 15 ␮M respectively. These results indicated delayed G1/S transition of cells in 16 h and S-phase arrest after 24 h treatment. The positive control resveratrol also demonstrated Sphase arrest (with 37.8% in S-phase and 53.4% cells in G1 phase at 24 h timepoint) as earlier reported (Fig. 4d and e). Furthermore, coagulin-L at concentration of 10 ␮M and 15 ␮M decreased MDI induced cell proliferation as estimated by 3 H-thymidine uptake (Fig. 4f).

To gain more insights about MCE arrest, various regulatory cell cycle proteins were analyzed including G1/S phase markers. When the preadipocytes were treated with coagulin-L containing MDI, the expression levels of CDK4, CDK2, and CDK6 were decreased at 16 h and 24 h. Expression of p27, the negative effector of the CDK regulatory proteins decreases during adipogenesis, but coagulin-L either increased or restored its expression (Fig. 5a). These observations suggested that coagulin-L might have induced cell cycle arrest in S-phase through the downregulation of CDK proteins and/or by preventing degradation of p27. Coagulin-L potentiate Wnt/ˇ-catenin pathway Fully confluent 3T3-L1 cells were treated with coagulin-L for 16 h and 24 h. The results revealed that, MDI induction leads to reduction of expression level of ␤-catenin, Wnt3a and LRP6 in 24 h. Addition of coagulin-L to MDI prevented this reduction (Fig. 5b). These results support a role of Wnt/␤-catenin pathway in coagulinL mediated adipogenic inhibition. Discussion In this study, we have shown anti-adipogenic potential of coagulin-L and its mechanistic insights using 3T3-L1 model system. Anti-adipogenic activity coagulin-L is also confirmed using other

M. Beg et al. / Phytomedicine 21 (2014) 406–414

411

Fig. 4. Inhibitory effect of coagulin-L on mitotic clonal expansion during the early stage of adipogenesis. (a) Differentiating 3T3-L1 and C3H10T1/2 were exposed to coagulinL for period mentioned in figure during adipogenesis, cells were stained and imaged as mentioned earlier. Lipid accumulation was measured in terms of extracted ORO quantified at 492 nM from (b) 3T3-L1 and (c) C3H10T1/2. (d) Time and concentration dependent effects of coagulin-L on cell cycle performed by flow cytometry as mentioned in methods. Resveratrol was used as positive control. (e) The percentage cell population in various cell cycle phases in coagulin-L exposed cells during MCE. (f) Coagulin-L concentration dependent reduction of [3 H]-thymidine uptake at 24 h in cells undergoing MCE during adipogenesis. Results expressed are mean ± SD of two exp. in triplicate with statistical significance *p < 0.05, **p < 0.01 and ***p < 0.001.

412

M. Beg et al. / Phytomedicine 21 (2014) 406–414

Fig. 5. Coagulin-L mediated effects on early phase signaling and cell cycle regulation. (a) Immunoblots exhibiting coagulin-L mediated expression levels changes (16 and 24 h) in cell cycle proteins CDK4, CDK2, CDK6 and P27 during MCE. (b) Same cell lysates were subjected to study relative expression levels of ␤-Catenin, Wnt3a, LRP6, components of Wnt/Catenin pathway. (c) Phosphorylation levels of C/EBP␤, mTOR and p70S6K and protein levels of CEBP␤ at 16 and 24 h after induction of MDI in absence and presence of 15 ␮M coagulin-L. (d) Comparative phosphorylation of AKT, ERK, mTOR, p70S6K and 4EBP in untreated and cogaulin-L treated confluent preadipocytes from 15 min to 2 h post MDI induction. ␤-actin was used as loading control. Experiments were repeated three times and representative blots were shown.

adipogenesis model systems, i.e., MDI induced murine stem cell line C3H10T1/2 and human MSCs. Mechanistic studies conclude coagulin-L modulates early signaling events, MCE and thus, leads to inhibition of adipogenesis. Adipogenesis program can be divided into early, intermediate, and late stages. All these stages are characterized by MDI induced potentiation of PI3K/AKT/mTOR pathway (Jung et al., 2013; Yu et al., 2008; Zhang et al., 2009), and its involvement is also evidenced by pharmacological or genetic interventional studies (Bell et al., 2000; Chang et al., 2009; El-Chaar et al., 2004). MCE is regulated through intricate orchestration of components of these pathways and phosphorylated C/EBP␤ (Tang et al., 2003). Berberine, Oleuropein and hydroxyl-tyrosol inhibit adipocyte differentiation by downregulation of PPAR␥, C/EBP␣ and its associated down-stream genes such as GLUT4, SREBP-1c, FAS, etc. (Drira et al., 2011; Huang et al., 2006). Coagulin-L exposure significantly inhibited PPAR␥, C/EBP␣ and GLUT4 expression at protein level in a time dependent manner during 2, 4 and 6 days (Fig. 3a). Since 4th day, is midway of adipogenic programming, we observed mRNA expression of downstream targets of PPAR␥ and found decrease in expression of FAS, LPL, SREBP-1c and aP2 (Fig. 3b). These evidences made us to investigate into downstream components of adipogenic signaling events, i.e., AKT/mTOR pathway. This pathway is involved in differentiation and protein synthesis, which is pre-requisite for cells undergoing adipogenesis. Although exact role of mTOR in adipogenesis is yet not clear, recently antiadipogenic activity of Cordycepin (3 -deoxyadenosine) as well as, Fisetin was reported to act by targeting mTOR and associated signaling (Jung et al., 2013; Takahashi et al., 2012). These findings establish the role of mTOR pathway along with C/EBP␤ and PPAR␥ in adipogenesis. Coagulin-L decreased phosphorylation of mTOR and its associated proteins P70S6K and 4EBP during adipogenesis (Fig. 5c and d). This suppression was observed as early as 2 h

of treatment and continued throughout the adipogenesis program. This was evident by immunobloting of 2, 16, 24 h and 2, 4 and 6 days protein samples (Figs. 3a, 5c and d). Thus, coagulin-L treatment, suggest potential involvement of mTOR-PPAR␥ pathway in coagulin-L induced adipogenesis inhibition. Epigallocatechin gallate (EGCG) and Matrine inhibit adipocyte differentiation by downregulating the ERK pathway (Kim and Sakamoto, 2012; Xing et al., 2010). Pigment epithelium-derived factor also inhibit adipogenesis by inhibiting MAPK/ERK signaling, subsequent induction of C/EBP␤ expression and its phosphorylation (Wang et al., 2009). Similarly, in our studies, we found coagulin-L mediated early MDI induced (15 min–2 h) ERK inhibition, followed by decreased C/EBP␤ phosphorylation at 16 h and 24 h timepoint between which MCE takes place (Figs. 4d, e and 5d). Many natural compounds that inhibit adipogenesis are capable of arresting cells in various cell cycle phases during MCE like resveratrol, picetanolol arrests cells in S-phase (Kwon et al., 2012a,b), and Genistein, Garcinol and Pterostilbene causes G2/M phase arrest (Hsu et al., 2012). Following to MDI induction, there is a rapid increase in cells population from G1 to S-phase in first 16 h, that finally enters into G2/M-phase by 24 h. Coagulin-L and resveratrol addition to MDI caused delay in G1/S-phase transition at 16 h timepoint in concentration dependent manner. Likewise, in later timepoint, i.e., 24 h post induction, S-phase population increases in concentration dependent manner, indicating delayed progression to G2/M. The MCE arrest trend was similar to earlier reported resveratrol, the positive control used in these studies (Kwon et al., 2012a,b) (Fig. 4d and e). MCE blockade was also confirmed by [3 H]-thymidine uptake assay. Coagulin-L inhibited MDI induced 3 H-thymidine incorporation in a concentration dependent manner (Fig. 4f). These studies indicate coagulin-L treatment lead to delayed G1 to S-phase transition, S-phase progression and MCE arrest. Since cell cycle regulatory proteins are involved in

M. Beg et al. / Phytomedicine 21 (2014) 406–414

coordinating MCE, it was important to explore coagulin-L mediated perturbations resulting into this changes. Cyclins and cyclin dependent kinases (CDKs) are involved in cell cycle regulation. In G1-phase Cyclin-D and Cyclin-E gets activated. Activated cyclin-D assembles with CDK4 or CDK6, while Cyclin E assembles with CDK2. In order to progress through S-phase, CDK2 and CDK1 need to be assembled with cyclin A and cyclin B. CDK2 is involved in phosphorylation of C/EBP␤, which in turn is required for transcriptional activation of PPAR␥ and C/EBP␣ during the early stage of adipocyte differentiation (Li et al., 2007). CDK inhibitor proteins like P21 and P27, required to be degraded for assemblies of Cyclin E and A to take place (Sherr, 1996). This allows growth arrested cells to make the G1/S transition, re-enter the cell cycle and proceed through the MCE during adipocyte differentiation (Choi et al., 2012). Previous Studies showed that disruption of CDK4 or activating mutations in CDK4 in primary mouse embryonic fibroblast results in reduced and increased adipogenesis, respectively, through modulation of PPAR␥ pathway (Abella et al., 2005). Interestingly, coagulin-L decreases CDK4, CDK6, CDK2 and stabilizes P27 levels (Fig. 5a). This indicates that in presence of coagulin-L, the requisite complex formation necessary for effective progression through S-phase may not have taken place and ultimately causes G1/S-phase transition delay and S-phase arrest. Wnts are secreted glycoproteins that signal through low density lipoprotein receptor-related protein (LRP) co-receptors. Canonical Wnt ligands, such as Wnt3a, are known to inhibit adipogenesis by stabilizing ␤-catenin and lead to repression of both C/EBP␤ and PPAR␥ (Lee et al., 2013). To assess involvement of this pathway, we treated 3T3-L1 cells with coagulin-L. This treatment lead to upregulation of expression of ␤-catenin, Wnt3a and LRP, which otherwise were downregulated following the adipogenic induction (Fig. 5b). Early phase overexpression of GATA2 and GATA3 are negatively regulate adipogenesis through downregulation of PPAR␥ and C/EBP␣ and is supported by many studies including Berberine (Hu and Davies, 2009; Tong et al., 2005). Coagulin-L treatment was found to cause significant increase in GATA2 expression and might be partially contributing to its antiadipogenic activity. Coagulin-L does not alter phosphorylation of AKT, but significantly decreased phosphorylation of its downstream target proteins like mTOR and P70S6K and 4EBP. It would be interesting but beyond the scope of this manuscript, to explore AKTindependent mechanistic regulation of phosphorylation changes in mTOR pathway mediated by coagulin-L. C/EBP␣ expression takes place after completion of MCE, considered to be anti-mitotic and negative modulator of MCE (Frith and Genever, 2008). Coagulin-L treatment increased early expression of C/EBP␣ and might also be contributing to MCE arrest by virtue of its anti-mitotic effects. Anti-adipogeneic compounds inhibiting differentiation of adipocyte leads to decreased number of adipocyte and does not necessarily be anti diabetic or insulin sensitizers. Recently there are concerns about anti-adipogenic compounds which may or may not be beneficial to overall health of person. Availability of fewer adipocyte had been proposed to cause insulin resistance and predispose to type II diabetes (Danforth, 2000; Stephens, 2012) On the contrary the classical notion is obesity leads to development of type 2 diabetes. Thiazolidine diones have been shown to increase adipogenesis and improve insulin sensitivity while type 2 diabetes is also represented by extreme lipoatrophic and morbid obesity. The other dual evolutionist hypothesis of selection of intermediary adipocyte genotype based on both insulin secretion and adipogenic potential put forward for explaining necessity of existence of adipocyte and limitations of its development (Ailhaud and Reach, 2001). Well known anti-adipogenic compounds, Curcumin and Berberine improve insulin sensitivity when given in vivo (Lee et al., 2006; Shao et al., 2012). Coagunolides have earlier been reported for modulating hepatic glucose metabolisms, decreases fasting blood glucose

413

levels and plasma insulin significantly and also it increases glucose tolerance in db/db mice. Further it has also been shown it normalizes concentration of plasma cholesterol, triglyceride, free fatty acids, low density lipoprotein and high density lipoprotein in the db/db mice (Maurya et al., 2008; Singh et al., 2012). Our present report shows that coagulin-L controls adipogenic differentiation, so taken together all indirect and direct evidences, the concerns regarding anti-adipogenic compounds causing insulin resistance does not apply in case of coagulin-L as it controls adipogenesis on one side while decreases circulating insulin and blood glucose in vivo. In summary, Withania Coagulans is described in Ayurveda for its use in diabetes like symptoms (Prameha). Withanolides are known for anti-hyperglycemic and anti-hyperlipidemic activity. This is first report of a withanolide, coagulin-L having anti-adipogenic activity. Coagulin-L modulates MDI mediated early phase events leading to MCE blockade by causing G1/S-phase transition, S-phase arrest and alteration of cell cycle specific proteins. In the late stage of adipogenesis it downregulates PPAR␥, C/EBP␣, the central regulators of adipogenesis. Furthermore mTOR pathway is also downregulated throughput adipogenesis, which might affect protein synthesis and overall differentiation machinery and subsequent biomass formation required for adipogenesis. Taken together in consideration of earlier reported biological activities, antiadipogenic activity reported herewith provides scientific basis for use of Withania Coagulans in preventive therapy of metabolic syndrome. Conflicts of interest The authors declare no conflict of interests. Acknowledgements Research work is supported by CSIR-CDRI Network project: “Towards holistic understanding of complex diseases: Unraveling the threads of complex disease (THUNDER)” Project No: BSC0102, and partially by Department of Biotechnology (DBT) project (GAP0079). MB is supported by SRF of DBT, PC is supported by JRF-CSIR, KS and SR are supported by JRF of University grant commission (UGC), New Delhi. Authors acknowledge FlowCytometry Facility of SAIF Division and technical support of Mr. Anoop K Srivastava of CSIR-CDRI. This manuscript bears CSIR-CDRI communication number: 8557. References Abella, A., Dubus, P., Malumbres, M., Rane, S.G., Kiyokawa, H., Sicard, A., Vignon, F., Langin, D., Barbacid, M., Fajas, L., 2005. Cdk4 promotes adipogenesis through PPARgamma activation. Cell Metab. 2, 239–249. Ahn, J., Lee, H., Kim, S., Ha, T., 2010. Curcumin-induced suppression of adipogenic differentiation is accompanied by activation of Wnt/beta-catenin signaling. Am. J. Physiol. Cell Physiol. 298, C1510–C1516. Ailhaud, G., Reach, G., 2001. Does obesity protect against diabetes? A new controversy. Ann. Endocrinol. (Paris) 62, S43–S54. Bell, A., Grunder, L., Sorisky, A., 2000. Rapamycin inhibits human adipocyte differentiation in primary culture. Obes. Res. 8, 249–254. Blanchard, P.G., Festuccia, W.T., Houde, V.P., St-Pierre, P., Brule, S., Turcotte, V., Cote, M., Bellmann, K., Marette, A., Deshaies, Y., 2012. Major involvement of mTOR in the PPARgamma-induced stimulation of adipose tissue lipid uptake and fat accretion. J. Lipid Res. 53, 1117–1125. Chang, G.R., Chiu, Y.S., Wu, Y.Y., Chen, W.Y., Liao, J.W., Chao, T.H., Mao, F.C., 2009. Rapamycin protects against high fat diet-induced obesity in C57BL/6J mice. J. Pharmacol. Sci. 109, 496–503. Choi, K.M., Lee, Y.S., Sin, D.M., Lee, S., Lee, M.K., Lee, Y.M., Hong, J.T., Yun, Y.P., Yoo, H.S., 2012. Sulforaphane inhibits mitotic clonal expansion during adipogenesis through cell cycle arrest. Obesity (Silver Spring) 20, 1365–1371. Chuang, C.C., Yang, R.S., Tsai, K.S., Ho, F.M., Liu, S.H., 2007. Hyperglycemia enhances adipogenic induction of lipid accumulation: involvement of extracellular signal-regulated protein kinase 1/2, phosphoinositide 3-kinase/Akt, and

414

M. Beg et al. / Phytomedicine 21 (2014) 406–414

peroxisome proliferator-activated receptor gamma signaling. Endocrinology 148, 4267–4275. Danforth Jr., E., 2000. Failure of adipocyte differentiation causes type II diabetes mellitus? Nat. Genet. 26, 13. Drira, R., Chen, S., Sakamoto, K., 2011. Oleuropein and hydroxytyrosol inhibit adipocyte differentiation in 3 T3-L1 cells. Life Sci. 89, 708–716. El-Chaar, D., Gagnon, A., Sorisky, A., 2004. Inhibition of insulin signaling and adipogenesis by rapamycin: effect on phosphorylation of p70 S6 kinase vs eIF4E-BP1. Int. J. Obes. Relat. Metab. Disord. 28, 191–198. Freise, C., Trowitzsch-Kienast, W., Erben, U., Seehofer, D., Kim, K.Y., Zeitz, M., Ruehl, M., Somasundaram, R., 2013. (+)-Episesamin inhibits adipogenesis and exerts anti-inflammatory effects in 3T3-L1 (pre)adipocytes by sustained Wnt signaling, down-regulation of PPARgamma and induction of iNOS. J. Nutr. Biochem. 24, 550–555. Frith, J., Genever, P., 2008. Transcriptional control of mesenchymal stem cell differentiation. Transfus. Med. Hemother. 35, 216–227. Hemalatha, S., Wahi, A.K., Singh, P.N., Chansouria, J.P., 2006. Hypolipidemic activity of aqueous extract of Withania coagulans Dunal in albino rats. Phytother. Res. 20, 614–617. Hsu, C.L., Lin, Y.J., Ho, C.T., Yen, G.C., 2012. Inhibitory effects of garcinol and pterostilbene on cell proliferation and adipogenesis in 3T3-L1 cells. Food Funct. 3, 49–57. Hu, Y., Davies, G.E., 2009. Berberine increases expression of GATA-2 and GATA-3 during inhibition of adipocyte differentiation. Phytomedicine 16, 864–873. Huang, C., Zhang, Y., Gong, Z., Sheng, X., Li, Z., Zhang, W., Qin, Y., 2006. Berberine inhibits 3T3-L1 adipocyte differentiation through the PPARgamma pathway. Biochem. Biophys. Res. Commun. 348, 571–578. Jung, C.H., Kim, H., Ahn, J., Jeon, T.I., Lee, D.H., Ha, T.Y., 2013. Fisetin regulates obesity by targeting mTORC1 signaling. J. Nutr. Biochem. 24, 1547–1554. Ka, S.O., Kim, K.A., Kwon, K.B., Park, J.W., Park, B.H., 2009. Silibinin attenuates adipogenesis in 3T3-L1 preadipocytes through a potential upregulation of the insig pathway. Int. J. Mol. Med. 23, 633–637. Kawai, M., Mushiake, S., Bessho, K., Murakami, M., Namba, N., Kokubu, C., Michigami, T., Ozono, K., 2007. Wnt/Lrp/beta-catenin signaling suppresses adipogenesis by inhibiting mutual activation of PPARgamma and C/EBPalpha. Biochem. Biophys. Res. Commun. 363, 276–282. Kim, H., Sakamoto, K., 2012. (−)-Epigallocatechin gallate suppresses adipocyte differentiation through the MEK/ERK and PI3K/Akt pathways. Cell Biol. Int. 36, 147–153. Kim, J.E., Chen, J., 2004. Regulation of peroxisome proliferator-activated receptorgamma activity by mammalian target of rapamycin and amino acids in adipogenesis. Diabetes 53, 2748–2756. Kwon, J.Y., Seo, S.G., Heo, Y.S., Yue, S., Cheng, J.X., Lee, K.W., Kim, K.H., 2012a. Piceatannol, natural polyphenolic stilbene, inhibits adipogenesis via modulation of mitotic clonal expansion and insulin receptor-dependent insulin signaling in early phase of differentiation. J. Biol. Chem. 287, 11566–11578. Kwon, J.Y., Seo, S.G., Yue, S., Cheng, J.X., Lee, K.W., Kim, K.H., 2012b. An inhibitory effect of resveratrol in the mitotic clonal expansion and insulin signaling pathway in the early phase of adipogenesis. Nutr. Res. 32, 607–616. Lee, H., Bae, S., Yoon, Y., 2013. The anti-adipogenic effects of (−)epigallocatechin gallate are dependent on the WNT/beta-catenin pathway. J. Nutr. Biochem. 24, 1232–1240. Lee, Y.S., Kim, W.S., Kim, K.H., Yoon, M.J., Cho, H.J., Shen, Y., Ye, J.M., Lee, C.H., Oh, W.K., Kim, C.T., Hohnen-Behrens, C., Gosby, A., Kraegen, E.W., James, D.E., Kim,

J.B., 2006. Berberine, a natural plant product, activates AMP-activated protein kinase with beneficial metabolic effects in diabetic and insulin-resistant states. Diabetes 55, 2256–2264. Li, X., Kim, J.W., Gronborg, M., Urlaub, H., Lane, M.D., Tang, Q.Q., 2007. Role of cdk2 in the sequential phosphorylation/activation of C/EBPbeta during adipocyte differentiation. Proc. Natl. Acad. Sci. USA 104, 11597–11602. Maurya, R., Akanksha, Jayendra, Singh, A.B., Srivastava, A.K., 2008. Coagulanolide, a withanolide from Withania coagulans fruits and antihyperglycemic activity. Bioorg. Med. Chem. Lett. 18, 6534–6537. Muoio, D.M., Newgard, C.B., 2006. Obesity-related derangements in metabolic regulation. Annu. Rev. Biochem. 75, 367–401. Rahman, Atta-Ur., Yousaf, M., Gul, W., Qureshi, S., Choudhary, M.I., Voelter, W., Hoff, A., Jens, F., Naz, A., 1998. Five New Withanolides from Withania coagulans. Heterocycles 48, 1801–1811. Rayalam, S., Della-Fera, M.A., Baile, C.A., 2008. Phytochemicals and regulation of the adipocyte life cycle. J. Nutr. Biochem. 19, 717–726. Shao, W., Yu, Z., Chiang, Y., Yang, Y., Chai, T., Foltz, W., Lu, H., Fantus, I.G., Jin, T., 2012. Curcumin prevents high fat diet induced insulin resistance and obesity via attenuating lipogenesis in liver and inflammatory pathway in adipocytes. PLoS ONE 7, e28784. Sherr, C.J., 1996. Cancer cell cycles. Science 274, 1672–1677. Singh, A.B., Singh, N., Akanksha, Jayendra, Maurya, R., Srivastava, A.K., 2012. Coagulanolide modulates hepatic glucose metabolism in C57BL/KsJ-db/db mice. Hum. Exp. Toxicol. 31, 1056–1065. Stein, C.J., Colditz, G.A., 2004. The epidemic of obesity. J. Clin. Endocrinol. Metab. 89, 2522–2525. Stephens, J.M., 2012. The fat controller: adipocyte development. PLoS Biol. 10, e1001436. Takahashi, S., Tamai, M., Nakajima, S., Kato, H., Johno, H., Nakamura, T., Kitamura, M., 2012. Blockade of adipocyte differentiation by cordycepin. Br. J. Pharmacol. 167, 561–575. Tang, Q.Q., Otto, T.C., Lane, M.D., 2003. Mitotic clonal expansion: a synchronous process required for adipogenesis. Proc. Natl. Acad. Sci. USA 100, 44–49. Tomiyama, K., Nishio, E., Watanabe, Y., 1999. Both wortmannin and simvastatin inhibit the adipogenesis in 3T3-L1 cells during the late phase of differentiation. Jpn. J. Pharmacol. 80, 375–378. Tong, Q., Tsai, J., Tan, G., Dalgin, G., Hotamisligil, G.S., 2005. Interaction between GATA and the C/EBP family of transcription factors is critical in GATA-mediated suppression of adipocyte differentiation. Mol. Cell. Biol. 25, 706–715. Wang, M., Wang, J.J., Li, J., Park, K., Qian, X., Ma, J.X., Zhang, S.X., 2009. Pigment epithelium-derived factor suppresses adipogenesis via inhibition of the MAPK/ERK pathway in 3T3-L1 preadipocytes. Am. J. Physiol. Endocrinol. Metab. 297, E1378–E1387. Xing, Y., Yan, F., Liu, Y., Liu, Y., Zhao, Y., 2010. Matrine inhibits 3T3-L1 preadipocyte differentiation associated with suppression of ERK1/2 phosphorylation. Biochem. Biophys. Res. Commun. 396, 691–695. Yu, W., Chen, Z., Zhang, J., Zhang, L., Ke, H., Huang, L., Peng, Y., Zhang, X., Li, S., Lahn, B.T., Xiang, A.P., 2008. Critical role of phosphoinositide 3-kinase cascade in adipogenesis of human mesenchymal stem cells. Mol. Cell. Biochem. 310, 11–18. Zhang, H.H., Huang, J., Duvel, K., Boback, B., Wu, S., Squillace, R.M., Wu, C.L., Manning, B.D., 2009. Insulin stimulates adipogenesis through the Akt-TSC2-mTORC1 pathway. PLoS ONE 4, e6189.