CYP1A2 contributes to alcohol-induced abnormal lipid metabolism through the PTEN/AKT/SREBP-1c pathway

https://doi.org/10.1016/j.bbrc.2019.04.034Get rights and content

Highlights

  • CYP1A2 is upregulated in L02 cells after 100 mM ethanol stimulated.

  • Inhibited CYP1A2 by siRNA-CYP1A2 or fluvoxamine ameliorates alcohol-induced lipid metabolism abnormality.

  • CYP1A2 regulated SREBP-1c positively, and PTEN negatively in ethanol treated L02 cells.

  • CYP1A2 participated in the abnormal lipid metabolism caused by alcohol stimulation through the PTEN/AKT/SREBP-1c pathway

Abstract

Abnormal liver lipid metabolism results in a series of liver diseases and contributes to tumourigenesis. The expression and activity of Cytochrome P450 1A2 (CYP1A2) is significantly increased in alcoholic fatty liver according to our previous studies. In this study, we aimed to explore the role of CYP1A2 in lipid metabolism abnormalities induced by alcohol and to investigate the underlying mechanisms. L02 cells were treated with siRNA-CYP1A2 or fluvoxamine and then stimulated with 100 mM ethanol for 24 h. The levels of ALT and TGs in the siRNA-CYP1A2 and fluvoxamine groups were significantly lower than those in the normal control group after ethanol treatment, and the expression of SREBP-1c was decreased when CYP1A2 was inhibited, suggesting that CYP1A2 may contribute to alcohol-induced irregular lipid metabolism by regulating sterol regulatory element-binding protein-1c (SREBP-1c). The expression of phosphatase and tensin homologue deleted on chromosome ten (PTEN) was decreased in normal cells, and it was further reduced when the expression of CYP1A2 was downregulated. Overexpression of PTEN led to decreased expression of p-AKT and SREBP-1c, which were elevated in both the normal and transfected groups after ethanol stimulation. In contrast, when PTEN was inhibited by siRNA-PTEN or bpv, the expression of PTEN was reduced in each group after ethanol treatment, while the expression of p-AKT and SREBP-1c was increased significantly. These results demonstrate that CYP1A2 may participate in the abnormal lipid metabolism caused by alcohol stimulation through the PTEN/AKT/SREBP-1c pathway.

Introduction

Given the increasing demand and consumption of ethanol-containing beverages in China in recent years, excessive alcohol intake has become one of the most important causes of liver injury. The initial stage of liver injury caused by persistent alcohol stimulation is lipid degeneration and fat accumulation, which strongly influence lipid metabolism. Lipid metabolism is one of the three major energy substance metabolic pathways in the human body and involves the metabolism of triglycerides (TGs), glycolipids, phospholipids, cholesterol and cholesterol esters.

Cytochrome P450 1A2 (CYP1A2) is a downstream gene of the aryl hydrocarbon receptor (AhR). Numerous studies have shown that there is a complex relationship between AhR and NF-κB [1,2]. It has been found that NF-κB can negatively regulate phosphatase and tensin homologue deleted on chromosome ten (PTEN) through many pathways. PTEN is the first tumour suppressor gene that has been found to possess dual phosphatase activity, and many tumour tissues present deficiency of PTEN [3]. Negative regulation of the phosphoinositide 3-kinases/protein kinase B (PI3K/AKT) pathway could be the biological function of PTEN. When this function is absent, AKT is overactivated, which may stimulate the proliferation and metastasis of tumour cells. There is evidence that the expression and activity of PTEN are decreased, the synthesis of fatty acid (FA) is increased, and the activity of AKT is elevated in mice treated with chronic ethanol [4]. The levels of TGs and alanine aminotransferase in the liver are increased tenfold and AKT activation is increased threefold in PTEN-KO mice fed ethanol persistently compared with normal control mice, suggesting that PTEN/AKT may contribute to lipid metabolism [5].

Recent research has found that activation of AKT could significantly enhance the expression of sterol regulatory element-binding protein-1c (SREBP-1c) [6,7], which is mainly expressed in the liver and is associated with FA biosynthesis. There is evidence that the expression of SREBP-1c in the livers of patients with fatty liver disease is fivefold higher than that in normal livers, and SREBP-1c is a key transcription factor regulating liver lipid synthesis [8]. Acetyl-CoA carboxylase (ACC1) and fatty acid synthase (FAS) are downstream target genes of SREBP-1c, both of which are essential enzymes for FA synthesis; mice with insufficient ACC1 and FAS die during the embryonic stage [9,10]. The expression of SREBP-1c is significantly increased in mice exposed to alcohol [11,12], the underlying mechanism of the regulation of SREBP-1c by alcohol is still unclear and may involve multiple signalling pathways. The specific purpose of this study was to explore whether the CYP1A2/PTEN/AKT/SREBP-1c pathway contributes to lipid metabolism in sustained ethanol-treated hepatocytes.

Section snippets

Cell culture and treatments

Human normal liver cells from the L02 cell line (Cell Bank of Chinese Academy of Sciences, Shanghai, China) were cultured in Dulbecco's modified Eagle medium (DMEM, HyClone, USA) supplemented with 10% foetal bovine serum (FBS, Gibco, USA) at 37 °C in a humidified atmosphere of 5% CO2. The L02 cells adhered overnight under these culture conditions and could be passaged after 24 h.

Methyl thiazolyl tetrazolium (MTT) assay

L02 cells were cultured in 96-well plates (1 × 104 cells/well) and then treated with or without ethanol at a range of

The viability of L02 cells treated with various concentrations of ethanol

The viability decreased after treatment with each concentration of ethanol for 12 h, but no clear toxicity was observed. L02 cells treated with ethanol ranging from 0 to 100 mM for 24 h did not show toxicity (Fig. 1A); however, in cells treated with concentrations ranging from 200 to 800 mM, cell proliferation was significantly inhibited. Stimulated with each concentration of ethanol for 48 h had an inhibitory impact on cells.

The stimulation of L02 cells by various concentrations of ethanol

The levels of ALT (Fig. 1B), AST (Fig. 1C) and TG (Fig. 1D) in cells

Discussion

The accumulation of lipids in hepatocytes caused by abnormal lipid metabolism is the initial step of alcohol-induced liver injury, which is a risk factor for alcoholic fatty liver and alcoholic hepatitis. Alcohol has been classified as a carcinogen by the International Agency for Cancer Research (IACR) of WHO, inappropriate intake of alcohol may induce a variety of cancers, including liver cancer [13,14]. Some articles about the phenomena and mechanisms of alcohol-induced carcinogenesis have

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