In this study, we investigated the anti-obesity and anti-hyperlipidemic mechanisms of lambertianic acid (LA) isolated fromPinus koraiensis leaves and the ethanol extract of Pinus koraiensis leaves (EPK), both in vitro and in vivo.
Differentiated 3T3L-1 cells were treated with EPK (25 or 50 μg/mL) or LA (200 μM) and analyzed by western blotting or RT-PCR. In vitro, lipid accumulation of adipocytes was observed using Oil-Red-O staining and triglyceride analysis. The contribution of AMPK to anti-obesity activity was assessed by siRNA-mediated AMPK knockdown. After AMPK silencing, expression of AMPK was observed by western blotting. To confirm the in vitro activity, an animal study was conducted by administering a normal diet, HFD, and EPK for 6 weeks. Obesity-related physiological parameters and protein levels were measured.
LA induced the expression of p-AMPK and inhibited PPARγ, C/EBP α, adiponectin, FAS, SREBP-1, and HMGCR expression. EPK containing LA significantly decreased lipid accumulation and triglyceride levels in the differentiated 3 T3-L1 cells. EPK treatment suppressed the expression of adipogenic transcription factors, FABP, GPDH, and cholesterol-synthesis-related factors in the differentiated 3 T3-L1 cells. EPK increased the expression of p-AMPK. The effects of EPK were reversed on inhibiting AMPK by using AMPK siRNA and compound C. In vivo analysis showed that body weight gain, serum triglyceride, total cholesterol, LDL cholesterol and AI value in the EPK treatment group were lower than those in the HFD control group. EPK induced the expression of p-AMPK and inhibited PPARγ in liver and adipose tissue.
Overall, the results suggest that EPK containing LA exerts significant anti-obesity and cholesterol-lowering effects by activating AMPK.
Obesity is a complex multifactorial chronic disease characterized by excess body fat and is associated with concurrent diseases that reduce life expectancy, including cardiovascular disease, stroke, hyperlipidemia, fatty liver, and diabetes [1,2]. Obesity caused by hypertrophy of adipose tissue as well as adipose tissue hyperplasia triggers the differentiation of preadipocytes into adipocytes . Adipocyte differentiation is regulated by crucial transcription factors such as peroxisome proliferator-activated receptor-γ (PPARγ) and CCAAT/enhancer-binding proteins α (C/EBPα). These transcription factors control the expression of many adipogenic proteins [4–7]. Several studies have reported that Sterol regulatory element-binding protein 1 (SREBP-1) is a transcription factor that regulates adipogenesis, cellular cholesterol, and cholesterol synthesis proteins, such as 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), in 3 T3-L1 adipocytes [8–10]. Adenosine monophosphate-activated protein kinase (AMPK) is a key enzyme in energy metabolism and is involved in regulation of glucose levels and lipid uptake. AMPK is expressed in a number of tissues, including adipose tissue, the liver, skeletal muscle, the heart, pancreatic beta cells, and brain cells [11, 12]. AMPK is phosphorylated and inactivates metabolic enzymes involved in fatty acid and cholesterol syntheses [13–15]. AMPK also provides an upstream signal of PPARγ/CEBPα and suppresses differentiation of preadipocytes into adipocytes [16–18]. Activation of AMPK decreases cellular cholesterol and fatty acids, such as SREBP-1 and HMGCR [19, 20]. Recently, the identification of a natural compound that can exert anti-obesity effects with fewer side effects than currently available prescription medications is attracting attention . One such compound is P. koraiensis (Korean nut pine), which is native to Korea, Japan, China, and Eastern Russia. The main chemicals in essential oil from P. koraiensis leaves (EOPK) are camphene, D-limonene, borneol, α-pinene, 3-carene, 4-carene, β-phellandrene, and fencyl . Our previous research has shown that EOPK has anti-hyperlipidemic , anti-diabetic , anti-obesity  and anti-cancer effects . P. koraiensis seed oil has been investigated to inhibition of lipid metabolism in rats and mice [25, 26]. However, the biological and biochemical effects of the ethanol extract of P. koraiensis (EPK) and its main compounds have not yet been proven. The EPK is easier to extract than EOPK. In addition, EPKs are convenient and easy to use. The purpose of this study is to investigate the anti-adipogenic effect of EPK on 3T3L-1 cells and the anti-obesity activity of EPK on high fat diet (HFD)-fed rats.