Effect of Aporosa villosa Stem Ethanolic Extract on Adipogenesis in 3T3-L1 Adipocytes

Obesity is responsible for the development of many diseases including hypertension, type 2 diabetes mellitus and atherosclerosis.1 Adipogenesis is the state of excess fat accumulation in adipocytes during the preadipocyte differentiation process2, and there is a study showing that increased adipocyte number related to obesity.3 Thus, the regulation of adipocyte number may be helpful for people who are obese. Several transcriptional factor genes such as CCAAT/enhancer-binding protein α (C/EBPα), peroxisome proliferator-activated receptor γ (PPARγ) and sterol regulatory element binding protein 1c (SREBP1c) are associated with the adipogenesis process.4 C/EBPα and PPARγ participate in a modulation of downstream lipogenic genes such as acetyl-CoA carboxylase (ACC), adipocyte fatty acid-binding protein 2 (aP2), cluster of differentiation (CD) 36, fatty acid synthase (FAS) and lipoprotein lipase (LPL).5


INTRODUCTION
Obesity is responsible for the development of many diseases including hypertension, type 2 diabetes mellitus and atherosclerosis. 1 Adipogenesis is the state of excess fat accumulation in adipocytes during the preadipocyte differentiation process 2 , and there is a study showing that increased adipocyte number related to obesity. 3 Thus, the regulation of adipocyte number may be helpful for people who are obese. Several transcriptional factor genes such as CCAAT/enhancer-binding protein α (C/EBPα), peroxisome proliferator-activated receptor γ (PPARγ) and sterol regulatory element binding protein 1c (SREBP1c) are associated with the adipogenesis process. 4 C/EBPα and PPARγ participate in a modulation of downstream lipogenic genes such as acetyl-CoA carboxylase (ACC), adipocyte fatty acid-binding protein 2 (aP2), cluster of differentiation (CD) 36, fatty acid synthase (FAS) and lipoprotein lipase (LPL). 5 Aporosa villosa is a plant that found in the Northern and Northeastern region of Thailand. It is used as a traditional medicine in some areas of Thailand, such as Ubon Ratchathani, for treating jaundice. However, this plant has limited pharmacological data. As obesity is an important condition that associated with the progression of several diseases, any novel substance or medicine, especially from natural sources, that can tackle this problem would become beneficial. Therefore, the present study interested to investigate whether Aporosa villosa extract has an anti-obesity action.
The 3T3-L1 adipocyte cells were used as a model for determination of Aporosa villosa stem ethanolic extract on adipogenesis process. This study may provide supportive data for future study in animal and human models.

Chemicals and reagents
Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/L glucose, sodium pyruvate and L-glutamine were obtained from Corning (Glendale, AZ, USA). Bovine calf serum (BCS), fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY, USA). 3T3-L1 adipocyte (ATCC CL-173) was purchased from American Type Culture Collection (Manassas, VA, USA). TRIzol reagent was obtained from Invitrogen (Carlsbad, CA, USA). High Capacity cDNA Reverse Transcription Kit was purchased from Applied Biosystems (Foster City, CA, USA). LightCycler  480 SYBR Green I Master was purchased from Roche Molecular Systems (Pleasanton, CA, USA). Other chemicals were obtained from Sigma-Aldrich (St. Louise, MO, USA).
The extracts were evaporated under reduced pressure to obtain the dry extracts. The yields of the dry powder were 8.22%.

Phytochemical screening
The crude ethanolic extracts of AS were tested for the presence of phenolic contents by using high-performance liquid chromatography with diode array detection and mass spectrometry detector (HPLC-DAD/MSD) method. 6 Caffeic acid, catechin, courmaric acid, ferulic acid, gallic acid, protocatechuic acid, quercetin, rutin, sinapic acid and vanillic acid were used as phenolic standards.

3T3-L1 adipocyte culture
The experiments of 3T3-L1 adipocytes were approved by the Thammasat University Institutional Biosafety Committee (TU-IBC 030/2562). The preadipocytes were plated in 24-well plates (2 × 10 5 cells/well), cultured in DMEM with 10% BCS, penicillin and streptomycin at 37°C in 5% CO 2 . After 2 days of cell confluence, the induction of cell differentiation was started by incubating in DMEM/10% FBS/penicillin/streptomycin with dexamethasone, 3-isobutyl-1-methylxanthine (IBMX) and insulin for 48 h. Then, cells were maintained and re-fed every 2 days with DMEM/10% FBS/penicillin/streptomycin/insulin. To determine the effect of AS on adipocyte differentiation, the cells were treated with AS at a concentration range of 3-100 μg/mL compared with the cells treated with dimethyl sulfoxide (DMSO) as a control group. Treatments of AS were started from the first day of the induction of cell differentiation (day 0) to the end of the experiment on day 8. Three independent experiments were performed, each in triplicate.

Oil Red O staining
At the end of the experimental period, cells were fixed with 10% formalin for 1 h and washed 3 times with phosphate-buffered saline (PBS). Then, cells were stained with 0.6% Oil Red O solution for 1 h and washed again with PBS 3 times. The stained cells were photographed by Primovert (Carl Zeiss, NY, USA) at ×200 magnification. After that, the stained Oil Red O was eluted with isopropanol and quantified by measuring absorbance at 540 nm.

Measurement of quantitative real-time PCR
Total RNA of 3T3-L1 cells was extracted using TRIzol reagent and reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit. The SYBR Green-based quantitative real-time PCR was performed using a LightCycler

Statistical analysis
Data were expressed as mean ± SEM. One-way analysis of variance (ANOVA) followed by Tukey's post-hoc test (Systat version 4.0, CA, USA) were used for investigating the significance difference among experimental groups. A statistical significance was set at P < 0.05.

Contents of phenolic compounds
Contents of phenolics were found in AS extract as shown in Table 1. The three main phenolics found in AS were rutin (199.20 μg/g), vanillic acid (106.91 μg/g) and catechin (98.60 μg/g).

Lipid accumulation
At day 8 of AS incubation, the microscopic observations of the Oil Red O staining showed that the AS at 3-100 μg/mL could inhibit lipid accumulation as compared to the untreated adipocyte group ( Figure  1A). The absorbance value revealed that the lipid accumulation in 3T3-L1 adipocytes was significantly reduced by AS extracts (3-100 μg/mL) compared with the untreated adipocyte group ( Figure 1B). Moreover, the inhibition of lipid accumulation of AS extracts did not cause cytotoxicity ( Figure 1C).

DISCUSSION/CONCLUSION
Obesity is a metabolic syndrome that is characterized by the excessive fat accumulation in adipose tissue. 8 The present study investigated the effect of Aporosa villosa stem ethanolic extract in 3T3-L1 adipocytes. AS extracts (3-100 μg/mL) were able to suppress lipid accumulation and adipogenic gene expressions in 3T3-L1 adipocytes model. Thus, inhibition of excessive lipid storage in adipocytes may have a positive effect in preventing obesity.
There are several stages in processing adipogenesis from preadipocytes to differentiated mature adipocytes. 9 In this study, AS (3-100 μg/mL) could inhibit lipid storage without showing cytotoxicity in adipocyte cells. There is a report indicating that the transcription factors C/EBPα, PPARγ and SREBP1c are key regulators for the differentiation of preadipocytes to mature adipocytes. 5 PPARγ is involved in the late stage of adipocytes differentiation, and its absence can block lipid droplet formation. [10][11] The present result showed that AS at the concentration of 10, 30 and 100 μg/mL could inhibit PPARγ gene in 3T3-L1 cells. It has been reported that C/EBPs and SREBPs are associated with regulating early stage of adipocyte differentiation process. 12    gene expressions of C/EBPα and SREBP1c were investigated in this study. The results showed that the AS treatment could reduce both genes in 3T3-L1 cells. These data demonstrated that AS treatment could inhibit the expression of transcription factor genes regulating adipogenesis.
The major transcription factors C/EBPα, PPARγ and SREBP1c involving in adipogenesis process regulate the adipocyte-specific markers such as LPL, FAS and CD36. 5 LPL is a major lipid synthesis enzyme which hydrolyzes lipoproteins from chylomicrons to free triglyceride in serum. 13 Overexpression of LPL is related to the initiation of lipid accumulation. 14 FAS is also a lipid synthesis enzyme. It can induce fatty acid synthesis and lipid accumulation. 15 CD36 plays a role in adipocyte cholesterol and adipogenesis metabolism. [16][17] Deficiency of CD36 could reduce the size and differentiation of adipocyte, and lipid accumulation in mice. 18 This study showed that AS extracts could decrease expression of these aforementioned genes. We speculate that AS inhibited the expressions of C/EBPα, PPARγ and SREBP1c genes, resulting in the suppression of the adipocyte-specific markers CD36, FAS and LPL genes in 3T3-L1 adipocytes.
Aporosa villosa is a plant that found in the Northern and Northeastern region of Thailand. Although it has been used in regional traditional medicinal practices, its systematic pharmacological data is limited. Generally, phenolic compounds widely found in plants possess antioxidant and anti-obesity activities. 19 In the present study, the AS extract was found to contain various phenolics, especially rutin, vanillic acid and catechin. These three main compounds have been previously reported to have an anti-obesity effect. [19][20][21] It is thus likely that these main phenolic compounds in the AS extract play a role in suppressing adipogenesis process in 3T3-L1 adipocytes by regulating C/EBPs/ PPARγ/SREBP1c signaling pathways.
In conclusion, this study shows the effect of AS on adipocyte differentiation and lipid accumulation. AS extract can inhibit adipogenesis process by decreasing the lipid accumulation and suppressing the expression of C/EBPα, PPARγ and SREBP1c genes and their downstream target genes CD36, FAS and LPL. Overall, these results suggest that AS has an anti-adipogenic activity in the 3T3-L1 adipocyte model, which implies that it may have potential as an antiobesity agent.