Novel Effect of p-Coumaric Acid on Hepatic Lipolysis: Inhibition of Hepatic Lipid-Droplets

Abstract

p-coumaric acid (p-CA), a common plant phenolic acid with multiple bioactivities, has a lipid-lowering effect. As a dietary polyphenol, its low toxicity, with the advantages of prophylactic and long-term administration, makes it a potential drug for prophylaxis and the treatment of nonalcoholic fatty liver disease (NAFLD). However, the mechanism by which it regulates lipid metabolism is still unclear. In this study, we studied the effect of p-CA on the down-regulation of accumulated lipids in vivo and in vitro. p-CA increased a number of lipase expressions, including hormone-sensitive lipase (HSL), monoacylglycerol lipase (MGL) and hepatic triglyceride lipase (HTGL), as well as the expression of genes related to fatty acid oxidation, including long-chain fatty acyl-CoA synthetase 1 (ACSL1), carnitine palmitoyltransferase-1 (CPT1), by activating peroxisome proliferator-activated receptor α, and γ (PPARα and γ). Furthermore, p-CA promoted adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) phosphorylation and enhanced the expression of the mammalian suppressor of Sec4 (MSS4), a critical protein that can inhibit lipid droplet growth. Thus, p-CA can decrease lipid accumulation and inhibit lipid droplet fusion, which are correlated with the enhancement of liver lipases and genes related to fatty acid oxidation as an activator of PPARs. Therefore, p-CA is capable of regulating lipid metabolism and is a potential therapeutic drug or health care product for hyperlipidemia and fatty liver.

Keywords: NAFLD; PPAR; hepatic lipase; lipid droplet; p-coumaric acid.

Figure 1

Effect of p-coumaric acid (p-CA) on the lipid metabolism of mice and HepG2 cells. (A): Total cholesterol (TC), total triglycerides (TG) in the blood and liver, and hepatic lipase activity of hyperlipidemic mouse induced by Triton WR1339 (Triton). The hyperlipidemic model was created using Triton WR1339 (300 mg/kg). p-CA was administered orally at the dose of 100 mg. (B,C): Lipid droplets under confocal microscope and the statistical data (n = 10–12 cells). The fluorescence intensity represents the integrated fluorescence intensity of oil red O staining per cell. (D): Cytotoxicity of p-CA in HepG2 cells according to MTT assay. (E): TC and TG in cells induced by oleic acid (OA). (F): TC and TG in cells induced by Triton RW1339. OA was used at 0.5 mM. Triton was administered at 20 μg/mL. p-CA was administered as 10 μg/mL in vitro. The data are presented as the mean ± S.D. from six mice or three independent experiments in vitro. VS. control groups, # p < 0.05 and ## p < 0.01. VS. hyperlipidemic mice or OA/Triton-treated cells in vitro, ** p < 0.01.

Figure 2

Effect of p-CA on the expression of some lipases in mouse liver in vivo and on HepG2 cells in vitro. (A): The expression of HTGL. (B): The expression of MGL. (C): The protein levels of p-HSL and HSL. Triton WR1339 was administered at 300 mg/kg in vivo and 20 μg/mL in HepG2 cells. Oleic acid (OA) was administered at 0.5 mM. The doses of p-CA were at 100 mg/kg in mouse model and 10 μg/mL in vitro. The symbol of + in red or black indicates how cells or mice were processed. The data are presented as the mean ± S.D. from six independent mice and three independent experiments in vitro. VS. control, # p < 0.05, ## p < 0.01. VS. the model mice (cells), * p < 0.05, ** p < 0.01.

Figure 3

Effect of p-CA on the MSS4 in mouse liver in vivo and HepG2 cells in vitro. (A): The expression of mRNA. (B): The expression of proteins. The symbol of + in red or black indicates how cells or mice were processed. The data are presented as the mean ± S.D. from six independent mice and three independent experiments in vitro. VS. control, # p < 0.05, ## p < 0.01. VS. the hyperlipidemic mice (cells), * p < 0.05, ** p < 0.01.

Figure 4

Effect of p-coumaric acid (p-CA) on lipid droplets in HepG2 cells with the proteins knocked down using shRNA. (A): Image of lipid droplets under a confocal microscope. (B): The statistical data of integrated fluorescence intensity per cell (n = 8–10 cells). The data are presented as the mean ± S.D. from three independent experiments. VS. control, ## p < 0.01. VS. OA groups, * p < 0.05, ** p < 0.01. NS: no significance.

Figure 5

Effect of p-CA on the expression of PKA and activation of AMPK in mouse liver in vivo and in HepG2 cells in vitro. (A): The expression of PKA. (B): The expression and activating phosphorylation of AMPKα at Thr172. (C): The effect of p-coumaric acid (p-CA) after the administration of an AMPK inhibitor, compound C (PubChem CID: 11524144, purchased from Selleck Chemicals, TX, USA). HepG2 cells were pre-treated with 7.5 μM of compound C for 2 h before Triton WR 1339 and p-CA. Then, the intracellular TG content was measured. The symbol of + in red or black indicates how cells or mice were processed. The data are presented as the mean ± S.D. from six independent mice and three independent experiments in vitro. VS. control, ## p < 0.01. vs. the model mice (cells), * p < 0.05, ** p < 0.01. NS: no significance.

Figure 6

Effect of p-CA on the PPARγ in mouse liver in vivo and in HepG2 cells in vitro. (A,B): The expression level of PPARγ. (C): TG in cells induced by Triton WR1339 treated with PPARγ inhibitor, GW9662. (D): TG in cells induced by Triton WR1339 when transfected with PPARγ shRNA plasmid. (E): The expression of PPARγ when knocked down by shRNA. (F): mRNA expression of MSS4, MGL, HSL and HTGL in cells induced by Triton WR1339, treated with or without GW9662. The symbol of + in red or black indicates how cells or mice were processed. The data are presented as the mean ± S.D. from six independent mice and three independent experiments in vitro. VS. the control, # p < 0.05, ## p < 0.01. VS. the hyperlipidemic mice (cells), * p < 0.05, ** p < 0.01. VS. the cells treated with Triton WR1339 and p-CA, $$ p < 0.01. NS: no significance.

Figure 7

The summary of p-CA on lipid metabolism. (A): The possible PPARγ-RXRa binding sites, with the promoters of the Rabif, Lipc, Lipe and Mgll genes as transcription factors, were predicted in the eukaryotic promoter database (EPD). These symbols indicated the distance between the predicted binding sites upstream within 1000 bp and the transcription start site in the gene promoter. (B): The relationship between PPARγ as a transcription factor and the promoters of the Rabif, Lipc, Lipe, Mgll, Cpt1a and Acsl1 genes was estimated using data from the CHIP-seq in the Cistrome Data Browser (DB). (C): The summary of the effect of p-CA on lipid metabolism.