Anti-adipogenic Constituents from Dioscorea opposita in 3T3-L1 Cells

Abstract

We previously reported the lipase inhibitory activity of the n-BuOH fraction of Dioscorea opposita (DOB) and its isolates. This study sought to evaluate their anti-adipogenic activity in terms of their effects on the adipogenic transcription factors peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) as well as phosphorylated AMP-activated protein kinase (p-AMPK) and carnitine palmitoyl transferase-1 (CPT-1). DOB apparently attenuated 3T3-L1 adipocyte differentiation (33.6% decrease at 20 µg/mL). In addition, a marked decrease (90.4%) in the expression of PPARγ was observed in the DOB-treated 3T3-L1 cells. Four isolates from DOB: (4E,6E)-1,7-bis(4-hydroxyphenyl)-4,6-heptadien-3-one (1), (3R,5R)-1,7-bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanediol (2), batatasin I (3), and (1E,4E,6E)-1,7-bis(4-hydroxyphenyl)-1,4,6-heptatrien-3-one (4), suppressed adipocyte differentiation by inhibiting PPARγ at 20 µM (85.9%, 68.6%, 76.2%, and 90.2% decrease, respectively) and C/EBPα (51.7%, 3.1%, 20.9%, and 59.8% decrease, respectively). Batatasin I was found to increase p-AMPK and CPT-1 at a concentration of 20 µM in 3T3-L1 adipocytes, resulting in inhibiting adipogenesis. Taken together, batatasin I might be responsible for the anti-adipogenic effect of DOB via inhibition of PPARγ and C/EBPα and activation of p-AMPK and CPT-1.

Development of obesity is characterized by an excessive adipocyte tissue mass.¹⁾ Adipose tissue growth results from both adipocytes proliferation and differentiation.²⁾ Adipocyte differentiation, also known as adipogenesis, is a complex process controlled by adipogenic transcription factors including peroxisome proliferator-activated receptor (PPAR) and CCAAT/enhancer binding protein (C/EBP).³⁾ PPARγ is considered to be a master regulator of adipogenesis in vivo and in vitro through transcription of various genes responsible for fat accumulation.^4–6) The cross-regulation of PPARγ and C/EBPα maintains adipocyte gene expression in the adipogenesis transcriptional pathway.⁷⁾

AMP-activated protein kinase (AMPK) is a major regulator of energy metabolism that effects on lipolysis molecules such as carnitine palmitoyl transferase-1 (CPT-1) and uncoupling protein-2 (UCP-2).⁸⁾ AMPK phophorylation suppresses the conversion of acetyl-CoA to malonyl-CoA, thereby inhibiting lipogenesis.^9,10) Furthermore, AMPK regulates the ligand-activated transcriptional factors such as PPARγ and C/EBPα, which lead to diminution in the fat cell growth.^11,12) Therefore, the activation of AMPK and the inhibition of PPARγ and C/EBPα in adipocytes are closely linked to the decrease of adipose tissue mass.^7,13)

Dioscorea opposita THUNB. (Dioscoreaceae) has been cultivated in China, Japan, and Korea as a food. It has been widely used for the treatment of gastrointestinal disorders.¹⁴⁾ The pharmacological properties of D. opposita extracts including anti-diabetic and neuroprotective effects have been reported earlier.^15,16) Besides, the D. opposita rhizomes contain diverse chemicals such as allantoin and batatasin, known to have antioxidant, anti-inflammatory, and glucose lowering activities.^17,18) However, there has not been any attempt to reveal the anti-adipogenic effect of D. opposita. Our recent research suggested that n-BuOH fraction of D. opposita (DOB) and its phenolic constituents possess inhibitory activities against pancreatic lipase in vitro.¹⁹⁾ This study was undertaken to evaluate the anti-adipogenic effect of certain constituents of DOB by measuring any change of PPARγ, C/EBPα, p-AMPK, and CPT-1 using 3T3-L1 cells.

MATERIALS AND METHODS

Chemicals

3-Isobutyl-1-methylxanthine (IBMX), dexamethasone, insulin, and (−)-epigallocatechin gallate (EGCG) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Dulbecco’s modified Eagle’s medium (DMEM), bovine calf serum (BCS), fetal bovine serum (FBS), and phosphate-buffered saline (PBS) were obtained from Thermo Scientific Hyclone (Franklin, MA, U.S.A.). Penicillin/streptomycin was from GIBCO (Grand Island, NY, U.S.A.) and 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) was purchased from Invitrogen (Grand Island, NY, U.S.A.). Specific antibodies recognizing PPARγ, C/EBPα, p-AMPK, and β-actin were obtained from Cell Signaling Technology (Danvers, MA, U.S.A.). Primary antibody against CPT-1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.).

Plant Extract and Compounds

The rhizomes of D. opposita THUNB. (Dioscoreaceae) were provided by Tong Yang Moolsan Co., Ltd. (Nonsan, South Korea), and identified by Prof. Gwang Jin Chang of the Korea National College of Agricultural and Fisheries. A voucher specimen (SNUPH-0822) has been deposited in the Medicinal Herb Garden, Seoul National University.

Fresh rhizomes of D. opposita (19 kg) were sliced into small pieces and lyophilized. The dried samples were extracted with 95% EtOH and partitioned with n-BuOH. Twenty-three compounds obtained from n-BuOH fraction of D. opposita (DOB) including (4E,6E)-1,7-bis(4-hydroxyphenyl)-4,6-heptadien-3-one (1), (3R,5R)-1,7-bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanediol (2), batatasin I (3), and (1E,4E,6E)-1,7-bis(4-hydroxyphenyl)-1,4,6-heptatrien-3-one (4)¹⁹⁾ (Fig. 1) were dissolved in dimethyl sulfoxide (DMSO) (0.1% v/v final concentration) and used for biological tests in 3T3-L1 cells.

Fig. 1. Chemical Structures of Active Compounds Isolated from DOB

Cell Culture and 3T3-L1 Cells Differentiation

Mouse embryo preadipocyte (3T3-L1) cell lines was obtained from ATCC (American Type Culture Collection, Manassas, VA, U.S.A.). Cells were cultured in DMEM containing 10% BCS, 100 U/mL penicillin and 100 µg/mL streptomycin in a humidified atmosphere of 5% CO₂ at 37°C. To induce differentiation, preadipocytes were seeded in 24-well plates (5×10⁴ cells/well) for Oil red O staining and 6-well plates (1×10⁵ cells/well) for Western blot analysis and cultured until confluent. Two days after confluency (day 0), adipocyte differentiation was induced with DMEM containing 10% FBS and hormone cocktail (0.5 mM IBMX, 1 µM dexamethasone, and 1 µg/mL insulin) in the presence of various concentrations of test samples (day 2). After 2 d, the medium was replaced with DMEM supplemented with 10% FBS containing 1 µg/mL insulin together with test samples (day 4). The medium was then maintained in 10% FBS/DMEM for additional 4 d (day 8). Extent of adipogenesis was determined in differentiated adipocytes by microscopy and measurement of lipid content as described below.

Oil Red O Staining

Differentiated adipocytes were stained with Oil red O using the method of Kasturi and Joshi with some modification.²⁰⁾ On day 8 after the induction of differentiation, cells were washed twice with PBS and fixed with 70% ethanol for 30 min. The cells were then stained with Oil red O solution (0.6% Oil red O in isopropanol : water=3 : 2) at room temperature for 1 h and washed twice with distilled water. The stained cells were visualized by Olympus IX50 microscope (Olympus, Tokyo, Japan).

Lipid Quantification in 3T3-L1 Adipocytes

Lipid accumulation was quantified with AdipoRed assay kit according to the manufacturer’s protocol (Lonza, Walkersville, MD, U.S.A.). AdipoRed is a Nile Red fluorescent reagent that enables the quantification of intracellular lipids from terminally differentiated adipocytes. Adipocytes were rinsed with PBS (400 µL) on day 8 after the induction of differentiation in presence of test samples. Subsequently, AdipoRed reagent (12 µL) and PBS (400 µL) were added to each well and cells were incubated for 10 min. The fluorescent intensity was measured at 485 nm (excitation) and 572 nm (emission). Percent lipid content was calculated in comparison with the vehicle control (100%).

Western Blot Analysis

Cells were harvested in cold PBS and lysed in lysis buffer (25 mM Tris–HCl, 1% NP-40, 1% sodium deoxycholate, 1% sodium dodecyl sulfate (SDS), 150 mM NaCl, 0.5 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1% protease inhibitor cocktail, pH 7.6). Lysates containing 30 µg protein were separated by 12% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% skim milk in TBST-buffer (Tween-20 in TBS, pH 7.2) and incubated with primary antibodies (anti-PPARγ, C/EBPα, p-AMPK, and CPT-1) (1 : 1000) at 4°C for 24 h. After washing three times in TBST, the membrane was incubated with secondary antibody (horseradish peroxidase-linked donkey antirabbit immunoglobulin G (IgG)) (1 : 1000) for 2 h. The result was visualized by using enhanced chemiluminescence (ECL) detection system.

Statistical Analysis

Data were analyzed by one-way ANOVA followed by Dunnett’s post hoc comparisons to determine the significant differences between treatment groups and the control group. Each value was presented as the mean±S.D. (n=3). The data were considered to be significant statistically if the probability had a value of 0.05 or less.

RESULTS AND DISCUSSION

Strategies for obesity treatment are classified into four categories such as reducing food intake, blocking nutrient absorption, increasing thermogenesis, and modulating fat metabolism/storage.^21,22) The inhibition of adipocyte differentiation is linked to the regulation of fat storage as well as metabolism. Hence, searching for new anti-adipogenic agents from natural sources might be a promising target for the prevention and treatment of obesity.^23,24)

DOB (1–20 µg/mL) dose-dependently inhibited fat accumulation during the differentiation of 3T3-L1 preadipocytes. The treatment of DOB suppressed 3T3-L1 adipogenesis by 33.5% of control at a concentration of 20 µg/mL (Fig. 2). The anti-adipogenic effects of twenty-three phenolics isolated from DOB, including structure moieties of dihydrostilbene, phenanthrene, dibenzoxepin, diarylheptanoid, and p-hydroxyphenylethyl-p-hydroxyphenyl propenoic acid, were evaluated on 3T3-L1 adipocytes. Four isolates, (4E,6E)-1,7-bis(4-hydroxyphenyl)-4,6-heptadien-3-one (1), (3R,5R)-3,5-dihydroxy-1,7-bis(4-hydroxyphenyl)-3,5-heptanediol (2), batatasin I (3), and (1E,4E,6E)-1,7-bis(4-hydroxyphenyl)-1,4,6-heptatrien-3-one (4), markedly attenuated adipocyte differentiation in the concentration range of 1–20 µM (Fig. 2). EGCG was used as a positive control since it has been known as potent inhibitor of adipocyte differentiation in 3T3-L1 cells, but it might be unstable under neutral or alkaline conditions.^25–27) Cell lipid content was distinctly decreased in adipocytes treated with 1, 2, 3, and 4 by 41.8%, 37%, 48.9%, and 38.1%, respectively (Fig. 2B). Especially, compound 3 (48.9% decrease) was the most effective in inhibiting adipogenesis at a concentration of 20 µM. Especially, batatasin I (48.9% decrease) was more effective than the EGCG-treated cells (45.6% decrease) at a concentration of 20 µM.

Fig. 2. Suppression of Adipocyte Differentiation by Compounds Isolated from DOB in 3T3-L1 Cells

Cells were exposed to differentiation cocktail (0.5 mM IBMX, 1 µM dexamethasone, and 1 µg/mL insulin) in the presence or absence of samples at different concentrations (1, 10, and 20 µM). After 8 d, morphological changes were detected by microscope (A) and lipid accumulations were measured by Nile Red fluorescent reagent (B). Representative result from three independent experiments was presented and data were expressed as the means±S.D. (n=3); ** p<0.01 and *** p<0.001 compared with the control

PPARγ and C/EBPα promote adipocyte differentiation by activating the transcription of genes involved in generating the adipocyte phenotype.²⁸⁾ In general, the occurrence of PPARγ is linked to the initiation of adipogenesis and C/EBPα is influential in maintaining the expression of PPARγ.⁷⁾ Medicinal plants, that specifically control PPARγ and C/EBPα expressions, have been emerged as targets for the treatment of obesity.²⁹⁾ Several natural polyphenols like catechin and procyanidin have been reported to down-regulate PPARγ and C/EBPα and have beneficial effects of suppressing obesity.^30,31) In the present study, transcriptional repressions of PPARγ and C/EBPα were detected by treatment of DOB. Exposure of DOB (20 µg/mL) to 3T3-L1 adipocytes resulted in down-regulation of PPARγ expression to 90.4% (Fig. 3). A considerable decrease in PPARγ protein level was observed in the compounds 1 (85.9% decrease), 2 (68.6% decrease), 3 (76.2% decrease), and 4 (90.2% decrease)-treated cells (Fig. 3A). Out of four compounds, 1 (51.7% decrease), 3 (20.9% decrease), and 4 (59.8% decrease) down-regulated expression of C/EBPα at 20 µM as shown in Fig. 3B.

Fig. 3. Inhibition of PPARγ (A) and C/EBPα (B) Expressions by D. opposita Compounds in Differentiated 3T3-L1 Cells

Cells were exposed to differentiation cocktail (0.5 mM IBMX, 1 µM dexamethasone and 1 µg/mL insulin) in the presence of DOB (20 µg/mL) or compounds (20 µM) for 8 d. The expression levels of PPARγ and C/EBPα were analyzed by Western blot. The content of PPARγ or C/EBPα in cell lysate was normalized with the content of β-actin. Representative result from three independent experiments was presented and data were expressed as the means±S.D. *p<0.05 compared with the control.

To reveal further details of the anti-adipogenic effect, the ability of four actives to suppress adipogenesis was determined using key enzymes like AMPK and CPT-1 involved in lipid metabolism in the cell culture system. Compounds 1–4 increased the activation of AMPK in 3T3-L1 adipocytes by phosphorylating the α-subunit of AMPK compared to corresponding controls (Fig. 4A). Treatments with 1 and 3 (20 µM) remarkably induced p-AMPK expression by 64% and 47%, respectively. In addition, time-dependent increase in p-AMPK and decrease in PPARγ were detected in 3T3-L1 adipocytes after incubation with 1 and 3 (20 µM) for 1–24 h (Fig. 5). It is well known that the activity of AMPK is inversely related to the expression of PPARγ and C/EBPα in adipogenesis.^32,33) AMPK inhibitors have been reported to stimulate adipocyte differentiation by up-regulating of PPARγ and C/EBPα in human adipose tissue-derived mesenchymal stem cells.³⁴⁾ However, direct relation between AMPK and adipogenic transcription factors remains unclear. According to our results, compounds 1–4 inhibited adipocyte differentiation by regulating AMPK as well as important markers of aipogenesis like PPARγ and C/EBPα at the cellular level. To conclude, down-regulation of PPARγ and C/EBPα by treatment with 1–4 might be partly mediated through AMPK, an upstream regulator of these proteins.

It is noteworthy that the anti-adipogenic effect of batatasin I (3) was mediated not only by the regulation of adipocyte specific transcription factors but also by modulating AMPK and CPT-1 expressions. CPT-1, one of the key enzymes involved in AMPK cascade, transfers cytosolic long-chain fatty acyl CoA into the mitochondria for fatty acid oxidation.⁸⁾ Impairment of CPT-1-mediated lipid oxidation is observed in obese patients and the inhibition of fat oxidation by CPT-1 inhibitor contribute to increase adiposity risk.^35,36) Naturally occurring AMPK activators such as curcumin and ginsenoside Rh2 augmented lipid metabolism by enhancing CPT-1 expression in 3T3-L1 cells.^37,38) Batatasin I (3) from DOB triggered the expression of CPT-1 to 169.1% at a concentration of 20 µM together with its AMPK stimulation (Fig. 4B). Treatment of 3T3-L1 adipocytes with batatasin I (3) time-dependently increased both p-AMPK and CPT-1 expressions (Fig. 5B). These data demonstrate that batatasin I (3) would contribute to the lipolysis by activating AMPK and CPT-1 as well as the anti-adipogenesis by inhibiting of PPARγ and C/EBPα in 3T3-L1 adipocytes.

Fig. 4. Activation of p-AMPK (A) and CPT-1 (B) Expressions in 3T3-L1 Adipocytes after Treatment with Isolates from DOB

Cells were exposed to differentiation cocktail (0.5 mM IBMX, 1 µM dexamethasone and 1 µg/mL insulin) in the presence of DOB (20 µg/mL) or compounds (20 µM) for 8 d. The expression levels of p-AMPK and CPT-1 were analyzed by Western blot. The content of p-AMPK or CPT-1 in cell lysate was normalized with the content of β-actin. Representative result from three independent experiments was presented and data were expressed as the means±S.D. **p<0.01 and ***p<0.001 compared with the control.

Fig. 5. Time–Course Analysis of p-AMPK, PPARγ, and CPT-1 Expressions in 3T3-L1 Cells Treated with Compounds 1 (A) and 3 (B)

3T3-L1 adipocytes were incubated with compound 1 (20 µM) or 3 (20 µM) for 1, 3, 6, 12, and 24 h, respectively. The expression levels of p-AMPK, PPARγ, and CPT-1 were analyzed by Western blot. Representative result from three independent experiments was presented.

This study provides in-depth understanding of the action of DOB and its constituents in the regulation of 3T3-L1 adipogenesis. Bioactive compounds 1–4 of D. opposita attenuated adipocyte differentiation by blocking major adipocyte marker proteins such as PPARγ and C/EBPα. In particular, the repression of PPARγ and C/EBPα in compounds 1–4-treated cells was partly caused by activation of AMPK. Batatasin I (3) possessed both AMPK and CPT-1 stimulation abilities in 3T3-L1 cells. Further study is warranted to fully elucidate the mechanism of anti-adipogenesis by D. opposita in animal adipose tissue.

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