Essential Oil of Pinus koraiensis Exerts Antiobesic and Hypolipidemic Activity via Inhibition of Peroxisome Proliferator-Activated Receptors Gamma Signaling

Our group previously reported that essential oil of Pinus koraiensis (EOPK) exerts antihyperlipidemic effects via upregulation of low-density lipoprotein receptor and inhibition of acyl-coenzyme A. In the present study, we investigated the antiobesity and hypolipidemic mechanism of EOPK using in vitro 3T3-L1 cells and in vivo HFD-fed rats. EOPK markedly suppressed fat accumulation and intracellular triglyceride associated with downregulation of adipogenic transcription factor expression, including PPARγ and CEBPα in the differentiated 3T3-L1 adipocytes. Additionally, EOPK attenuated the expression levels of FABP and GPDH as target genes of PPARγ during adipocyte differentiation. Furthermore, PPARγ inhibitor GW9662 enhanced the decreased expression of FABP and PPARγ and fat accumulation induced by EOPK. To confirm the in vitro activity of EOPK, animal study was performed by administering normal diet, HFD, and/or EOPK at the dose of 100 or 200 mg/kg for 6 weeks. Consistently, EOPK significantly suppressed body weight gain, serum triglyceride, total cholesterol, LDL cholesterol, and AI value and increased HDL cholesterol in a dose-dependent manner. Immunohistochemistry revealed that EOPK treatment abrogated the expression of PPARγ in the liver tissue sections of EOPK-treated rats. Taken together, our findings suggest that EOPK has the antiobesic and hypolipidemic potential via inhibition of PPARγ-related signaling.


Introduction
Obesity as a medical condition of excess body fat caused by an accumulation of adipose tissue mass [1] is tightly associated with various health disorders such as hyperlipidemia, diabetes, and cardiovascular diseases [2]. In obesity, adipocytes accumulate excessive fat and adipocytokine production is disrupted [3,4]. Adipose tissue development observed in obese individuals is closely related to hypertrophy and hyperplasia, the latter involving proliferation and differentiation of preadipocytes to adipocytes. The peroxisome proliferatoractivated receptor (PPAR) and CCAAT/enhancer-binding proteins (C/EBP) families of transcription factors regulate this adipocyte differentiation [5][6][7][8]. These transcriptional factors regulate the expression of genes involved in the induction adipocyte phenotypes [5].
Pharmaceutical antiobesity drugs are generally developed to loss or control body weight. Although various prescribed or nonprescribed medications have been used for treatment of obesity patients, there is a limitation to apply long-term use because of their severe side effects. Only orlistat (Xenical) has been approved by the FDA for long-term use. Recently, many studies reported antiobesity activity of natural products and suggested the potential as antiobesity agents or their supplements.
Recently, many studies reported that natural products such as Rhizoma Polygonati falcatum (RPF) [14], Boussingaultia gracilis Miers var. pseudobaselloides Bailey [15], Chinese black tea (Pu-erh tea) extract, and gallic acid [16] showed the potential of antiobesity activity in vitro or in vivo. In the present study, we investigated the antiobesic and hypolipidemic effect of essential oil of P. koraiensis SIEB (EOPK) in 3T3-L1 cells by Oil-Red O staining, measuring triglyceride, and analyzing expression levels of adipogenic factors and in high-fat diet-fed rats by measuring body weights and fats, and lipid metabolites.

Preparation of Essential Oil of P. koraiensis Leaves (EOPK).
EOPK was prepared using the hydrodistillation method [17]. Driedand pulverized P. koraiensis leaves were immersed in distilled water and submitted to steam distillation using an apparatus with a condenser (Hanil Labtech, Seoul, Korea). The distillation continued for 3-4 h at 90 ∘ C, and then the volatile compounds contained in the water-soluble fraction were allowed to settle for 20 min. The essential oil layer was separated and purified through microfiltration.

Cell
Culture. 3T3-L1 preadipocytes were purchased from Korean Cell Line Bank (KCLB). The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) with 10% FBS in a humidified atmosphere of 95% air and 5% CO 2 at 37 ∘ C.

Differentiation Induction and Oil-Red O Staining.
The preadipocyte 3T3-L1 cells were plated onto 6-well plates on day 0 and incubated to confluent status. For adipocyte differentiation, the confluent cells were treated with 1 M dexamethasone, 1 g/mL insulin, and 0.5 mM IBMX for 2 days, and the medium was replaced by fresh normal medium only containing 1 g/mL insulin. On day 8, the differentiated adipocyte cells were cultured in the presence or absence of EOPK (100 g/mL) for 2 days. The cells were fixed with 2% paraformaldehyde, washed twice with PBS, and finally stained with Oil-Red. After dissolving, cellular-lipid retained Oil-Red O in isopropanol, and adipocyte expression was estimated by measuring OD using microplate reader (Sunrise, TECAN, Mannedorf, Switzerland) at 510 nm.
The blots were washed, exposed to horseradish peroxidase-(HRP-) conjugated secondary antibodies for 2 h, and finally examined by enhanced chemiluminescence (ECL) (GE Health Care Bio-Sciences, Piscataway, NJ, USA).

Animals, Diet Manipulation, and EOPK Treatment. Male
Sprague-Dawley rats, at 4 weeks of age, were purchased from Hyo-Chang Science (Daegu, Korea) and maintained under conventional conditions. Fifty rats were divided into four groups; normal group (low fat diet), control group (high-fat diet), two EOPK-treated groups-and atorvastatin (positive control) treated group consuming high-fat diets (10 rats per group). Rats were fed the normal (low fat) diet (group 1) or the high-fat diet (groups 2-5) for 6 weeks. High-fat diet composition was described in Table 1. For EOPK treatment, EOPK dissolved in 4% tween 80/normal saline was orally administered once daily to the rats at the doses of 100 (group 3) and 200 mg/kg (group 4) for 6 weeks from the 1st day of high-fat diet feeding, whereas PBS was orally administered to the rats in control group (group 2). As a positive control, atorvastatin was administered at a dose of 10 mg/kg (group 5).

Preparation of Rat Serum.
Whole blood was collected from rats by cardiac puncture method, and serum was isolated by centrifugation at 3000 rpm for 10 min.

Effect of EOPK on Fat Accumulation in 3T3-L1
Adipocyte-Like Cells. Cytotoxicity of EOPK was determined by MTT assay in 3T3-L1 preadipocytes. Cells were treated with various concentrations of EOPK (0, 12.5, 25, or 50 g/mL) for 24 h. As shown in Figure 1(a), EOPK had no significant effect on the viability of 3T3-L1 cells. To investigate whether EOPK can affect the cellular differentiation into adipocytes, Oil-Red O staining was performed in 3T3-L1 cells treated with EOPK for 8 days. The differentiated 3T3-L1 adipocytes significantly increased fat accumulation and intracellular triglyceride (Figures 1(b), 1(c), and 1(d)), when compared to preadipocytes. The fat deposits were decreased by 57 and 78% at the treatment with various concentrations of EOPK (25 or 50 g/mL), respectively, compared to untreated control, adipocytes. Results indicate that 50 g/mL of EOPK was the most effective ability to inhibit adipocyte differentiation (Figures 1(b) and 1(c)). EOPK treatment reduced the level of triglyceride in cell in a dose-dependent manner (37 and 60% at concentrations of 25 and 50 g/mL, resp.), compared to preadipocytes (80.04%) (Figure 1(d)).

Effect of EOPK on the Expression of C/EBP, PPAR , GPDH,
and FABP during the Differentiation of Adipocytes. PPAR , C/EBP , GPDH, and FABP are key factors involved in adipogenesis. In particular, PPAR controls adipogenic factors as a key transcription factor during adipocyte differentiation. Both mRNA and protein levels of PPAR , C/EBP , and GPDH were downregulated by EOPK in a dose-dependent manner (Figures 2(a) and 2(b)). To elucidate the underlying mechanism of EOPK, PPAR antagonist GW9662 was used in 3T3-L1 adipocytes. PPAR inhibitor GW9662 enhanced the decreased expression of FABP and PPAR by EOPK (Figure 2(c)).
As shown in Figures 2(d) and 2(e), the lipid accumulation in 3T3-L1 adipocytes treated with EOPK and GW9662 was significantly reduced compared to untreated control. Consistently, immunohistochemistry revealed that EOPK treatment attenuated the expression of PPAR in the liver tissue sections of EOPK-treated group (100 and 200 mg/kg) compared to untreated control group (Figure 3(c)). Taken together, these results indicate that EOPK inhibits adipocyte differentiation via suppressing PPAR activation.

Effect of EOPK on Body Weight of High-Fat Diet-Fed Rats.
The body weights of normal (group 1), control (group 2), EOPK-treated groups (groups 3 and 4), and atorvastatintreated group (group 5) were monitored once every week for 6 weeks. As shown in Figure 3(a), the body weight of high-fat fed control group was significantly increased 2 weeks after feeding compared to normal low fat group. In contrast, the body weight gain was dose dependently abrogated in EOPK-treated groups, from the date of 3-week treatment of EOPK. Also, six weeks after treatment, the body weight was significantly gained to 331.4 ± 2.8 g in high-fat fed control group compared to normal group (281.6 ± 1.7 g). However, EOPK significantly suppressed the body weights to 309.5 ± 1.8 g and 303.7 ± 2.0 g, respectively, at doses of 100 mg/kg and 200 mg/kg, almost coming up with atorvastatin as positive control (296.5 ± 1.9 g).

Effects of EOPK on Abdominal Fat Weight of High-
Fat Diet-Fed Rats. The retroperitoneal and epididymal fat weight was measured to test whether the body weight normalization by EOPK in high-fat diet-fed rats is associated with a reduction of fat content in body. In the present study, as expected, high-fat diet significantly increased the retroperitoneal fat weight to 18.3 ± 2.91 mg/g from 6.21 ± 1.26 mg/kg body weight. In contrast, EOPK decreased the retroperitoneal fat weight to 15.7±1.86 and 12.7±1.43 mg/g at doses of 100 and 200 mg/kg, respectively, compared to control group (Figure 3(b)). Likewise, epididymal fat weight was also increased in the high-fat diet-fed rats compared to normal group. Oral administration of EOPK decreased epididymal fat pad weight by 10.9 ± 1.85 and 8.52 ± 1.96 mg/g at doses of 100 and 200 mg/kg, respectively (Figure 3(b)).

Effects of EOPK on Serum Lipid and Cholesterol Levels in High-Fat Diet-Fed Rats.
The consumption of the high-fat diet significantly increased triglyceride in control group compared to the normal low fat group (Figure 4(a)). EOPK treatment decreased level of serum triglyceride from 131.8 ± 18.2 to 109.3 ± 11.5 and 94.2 ± 8.43 mg/dL at 100 and 200 mg/kg, respectively (Figure 4(a)). Additionally, the consumption of the high-fat diet significantly increased serum total cholesterol compared to the normal low fat group, while EOPK significantly reduced the level of total cholesterol in a dosedependent manner (Figure 4(b)). The intake of the highfat diet significantly decreased level of HDL but increased level of LDL compared to normal group (Figure 4(c) control). However, EOPK did not have a significant effect on LDL while elevating HDL level in a dose-dependent manner compared to the high-fat diet control group (Figure 4(c)). Consistently, EOPK treatment significantly decreased the atherosclerosis index (AI) value in a dose-dependent manner compared to the high-fat diet control (Figure 4(d)).

Effects of EOPK on Hepatic Triglyceride and Cholesterol
in High-Fat Diet-Fed Rats. The consumption of the high-fat diet significantly increased hepatic triglyceride ( Figure 5(a)) and total cholesterol ( Figure 5(b)), when compared to the normal low fat group. Oral treatment with EOPK reduced the level of triglyceride from the liver in a dose-dependent manner (24.4 ± 2.47 and 21.2 ± 1.88 mg/g at doses of 100 and 200 mg/kg, resp.), compared to control group (29.8 ± 3.25 mg/g tissue) ( Figure 5(a)). In addition, EOPK administration significantly lowered total cholesterol level in liver from 16.9 ± 2.18 mg/g in the high-fat diet control group to 12.6 ± 1.42 and 10.4 ± 1.59 mg/g at 100 and 200 mg/kg, respectively ( Figure 5(b)).

Discussion
The Korean pine (P. koraiensis) is an important afforestation trees in Korea and also distributed in China, Russia, Japan, and Europe [21]. P. koraiensis extract attenuated the increase in blood pressure in spontaneously hypertensive rats [13], and its constituent pinolenic acid had cholesterollowering effect via regulation of LDL receptor activity in HepG2 hepatoma cells [22]. In addition, P. koraiensis nut oil effectively regulated satiety hormones and prospective food intake in postmenopausal overweight women [11]. Our group also reported that essential oil from P. koraiensis leaves had antihyperlipidemic effects via upregulation of LDL receptor and inhibition of acyl-coenzyme A: cholesterol acyltransferase [9]. In the current study, we demonstrate that EOPK has antiobesity effects in 3T3-L1 adipocytes and high-fat dietfed rat models. Adipogenesis is the process of differentiation by which undifferentiated preadipocytes are converted into differentiated adipocytes [23] and subsequently mediate the synthesis and accumulation of lipid [24]. 3T3-L1 cells are the best characterized model to study adipogenesis in vitro [25]. Oil-Red O staining revealed that EOPK significantly suppressed fat accumulation and intracellular triglyceride and decreased expression of PPAR , CEBP , FABP, and GPDH in the differentiated 3T3-L1 adipocytes with no cytotoxicity, indicating the inhibitory effect of EOPK on adipocyte differentiation. Similarly, many natural compounds, including EGCG, berberine, and curcumin, suppressed PPAR signaling, since PPAR antagonists have been reported to effectively inhibit adipogenesis and improve insulin sensitivity in vitro and in vivo [26]. Furthermore, here the antiobesic effect of P. koraiensis was confirmed in high-fat diet-(HFD-) treated SD rats at the doses of 100 or 200 mg/kg via blockage of body weight gain. Since loss of body weight is mainly associated with the decrease of fat pad mass as a result of reduction of adipocyte size or triglyceride accumulation [27], our data that EOPK significantly reduced the retroperitoneal and epididymal fat weight and also serum triglyceride compared to HFD fed rats suggest that EOPK can block obesity via inhibition of lipid metabolism including triglyceride.
Cholesterol is also an important component of fat complex. In particular, low level of serum HDL cholesterol is tightly linked to the occurrence of obesity [28]. In our study, administration of EOPK significantly abrogated the contents of total cholesterol in serum. In addition, EOPK significantly increased level of HDL cholesterol in a dose-dependent manner compared to HFD control group but not LDL cholesterol. Lipid parameters in the blood can be affected Evidence-Based Complementary and Alternative Medicine 9 by the hepatic metabolisms. As expected, we observed that the hepatic contents of triglyceride and cholesterol were significantly escalated in HFD fed rats compared to normal control. In contrast, EOPK treatment significantly lowered the contents of triglyceride and total cholesterol in the liver, implying that EOPK regulates lipid metabolism including triglyceride and cholesterol against obesity.
In summary, EOPK inhibited fat accumulation, intracellular triglyceride, and expression of PPAR , CEBP , FABP, and GPDH in the differentiated 3T3-L1 adipocytes. Also, EOPK attenuated serum and hepatic contents of triglyceride and total cholesterol in HFD fed rat models. Furthermore, EOPK decreased the expression of PPAR in the liver tissue sections of EOPK-treated rats. Overall, our findings suggest the potential of EOPK as an antiobesity agent.