Ethanol-mediated upregulation of APOA1 gene expression in HepG2 cells is independent of de novo lipid biosynthesis

Abstract

Background: Moderate alcohol intake in human increases HDL-cholesterol, and has protective effects against cardiovascular disease (CVD). Although de novo lipid synthesis inhibitors are highly effective in lowering total and LDL-cholesterol they have only modest effects on raising HDL-C. A better understanding of the mechanism of ethanol-mediated HDL-C regulation could suggest new therapeutic approaches for CVD.

Methods: Human hepatoblastoma (HepG2) and colorectal epithelial adenocarcinoma (Caco-2) cells were incubated in the presence of varying concentrations of ethanol in the culture medium, with or without addition of de novo lipid synthesis (DNLS) inhibitors (atorvastatin and/or TOFA). ApoA1 protein was measured by Western blot, and RNA of lipid pathway genes APOA1, APOC3, APOA4, APOB100, HMGCR, LDLR, and SREBF2 by quantitative RT-PCR. Lipoproteins (VLDL, LDL, and HDL) and lipids were also monitored.

Results: Ethanol stimulated ApoA1 protein (both cytoplasmic and secreted) and APOA1 RNA levels in HepG2 cells in a dose sensitive way, with ~ 50% upregulation at 100 mM ethanol in the medium. The effect was not observed in intestinal-derived Caco-2 cells. DNLS inhibitors did not block the upregulation of ApoA1 RNA by ethanol; TOFA alone produced a modest increase in ApoA1 RNA. Ethanol had no effect on ABCA1 protein levels. Addition of ethanol to the cell medium led to modest increases in de novo synthesis of total cholesterol, cholesteryl esters and triglycerides, and as expected these increases were blocked when the lipid synthesis inhibitors were added. Ethanol stimulated a small increase in HDL and VLDL but not LDL synthesis. Ethanol in the cell medium also induced modest but measurable increases in the RNA of APOC3, APOA4, APOB, LDLR, and HMGCR genes. Unlike APOA1, induction of RNA from APOC3 and APOA4 was also observed in Caco-2 cells as well as HepG2 cells.

Conclusion: This study has verified the previously reported upregulation of APOA1 by exposure of HepG2, but not Caco-2 cells, to ethanol in the culture medium. It is shown for the first time that the effect is dependent on RNA polymerase II-mediated transcription, but not on de novo biosynthesis of cholesterol or fatty acids, and therefore is not a generalized metabolic response to ethanol exposure. Some other lipid pathway genes are also modulated by ethanol exposure of cells. The results reported here suggest that the proximal signaling molecule leading to increased APOA1 gene expression in response to ethanol exposure may be free acetate or acetyl-CoA.

Take home: Upregulation of ApoA1 gene expression in hepatoma cells in culture, upon exposure to moderate ethanol concentrations in the medium, occurs at the level of RNA and is not dependent on new cholesterol or fatty acid synthesis. The primary signaling molecule may be free acetate or acetyl-CoA. These results are important for understanding the mechanism by which moderate alcohol consumption leads to upregulation of serum HDL-cholesterol in humans, and suggests new approaches to targeting HDL as a risk factor for cardiovascular disease.

Keywords: APOA1; Alcohol; Apolipoprotein A1; Cardiovascular disease; Ethanol; HDL; HEPG2; Liver.

Fig. 1

ApoA1 and ABCA1 protein expression in cells treated with ethanol or acetate. HepG2 cells were incubated in standard medium with addition of the indicated concentration of ethanol (0, 10, 25, 50, 100 mM). Cell lysates (panel a) or TCA-precipitated culture medium (panel b) was analyzed by Western blot to detect ApoA1 protein. Actin protein was used to control for loading of cytoplasmic extracts, equivalent volumes were precipitated to control for loading of protein precipitated from the culture medium. In all cases plates contained similar densities of cells. c Quantified cytoplasmic ApoA1 protein relative to β-actin averaged from three independent experiments. d Quantified precipitated ApoA1 protein from culture mediums, averaged from three independent experiments. e HepG2 cells were treated with the indicated concentrations of ethanol for 24 h, and cell extract were ABCA1 protein was assayed by Western blot. f HepG2 cells were treated with the indicated concentrations of ethanol or sodium acetate for 24 h, and cell extract ApoA1 protein was assayed by Western blot. Data are expressed as means ± SD of three experiments for ApoA1 protein and RNA expression (1 way ANOVA with multiple comparisons; *P < 0.05, **P < 0.01, ***P < 0.001 vs. control

Fig. 2

APOA1 RNA is upregulated by ethanol and requires RNA polymerase II activity. Relative RNA expression levels were determined by RT-qPCR as described in Methods. HepG2 were cultured to ~ 80% confluence on six well plates before treating with ethanol. a HepG2 cells were exposed to 100 mM ethanol and/or α-amanitin as per Materials and Methods. b and c Relative RNA expression levels were determined by RT-PCR as described in Methods. Dose response curve of APOA1 RNA at the indicated final concentrations of ethanol in the medium. The error bars represent the SD from the mean of five assays of an individual experiment. *Significant difference, ethanol treatment versus untreated control (1 way ANOVA with multiple comparisons, n = 5; *P < 0.05, **P < 0.01, ***P < 0.001 vs. control)

Fig. 3

Effects of DNLS inhibitors on induction of APOA1 RNA expression and on cellular lipid concentration in cells treated with ethanol. Relative RNA expression levels were determined by RT-qPCR as described in Methods. a and b Expression of APOA1, LDLR, HMGCR and SREBF2 in HepG2 cells cultured in LPDS medium and treated at the indicated final concentration of ethanol (100 mM) and/or atorvastatin (20uM). c HepG2 cells were cultured in standard medium, and exposed to combinations of 100 mM ethanaol, 20 uM atorvastatin, and/or 20 uM TOFA as per Materials and Methods. The error bars represent the SD from the mean of five assays of an individual experiment. *Significant difference, ethanol treatment versus untreated control (1 way ANOVA with multiple comparisons, n = 5; *P < 0.05, **P < 0.01, ***P < 0.001 vs. control). For the effect of DNLS Inhibitors on cellular lipid concentration. HepG2 cells were cultured 21 days in DMEM+ 10% FBS for differentiation, after O/N starvation, cells were incubated with [¹⁴C] oleic acid and treated with atorvastatin (20 uM) or ETOH (100 m M) + Atorvastatin (20 mM) for 24 h (d and e). For the f cells were treated with TOFA (20 uM) or ETOH (100 m M) + TOFA (20 mM) for 24 h with the concentration indicated of ETOH. After the incubation, lipids were extracted, separated by thin-layer chromatography and quantified as described in Materials and Methods. Data were analyzed as dpm/mg of total protein and represent means±SD for n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control

Fig. 4

Effect of ETOH on lipoproteins VLDL, LDL and HDL output by HepG2 cells. Following 21 days of differentiation, cells were incubated with [¹⁴C] oleic acid and ETOH (0, 50, and 100 mM) for 20 h. VLDL, LDL and HDL were isolated by ultracentrifugation according to their specific densities. Radioactivity incorporated into each fractions was further determined. Data were analyzed as dpm/mg of total protein and represent means±SD for n = 3 independent experiments. *P < 0.05, **P < 0.01 vs control

Fig. 5

Effects of ethanol on expression of genes involve in the cholesterol biosynthesis pathway. RNA levels of the indicated genes were determined by RT-qPCR as described in Methods. a, bAPOC3 and APOA4 RNA levels were compared between cell types HepG2 and Caco-2 respectively, treated with the indicated final concentrations of ethanol. c, dAPOC3, APOA4 and APOB gene expression were compared between standard medium (FBS) and lipid-depleted medium (LPDS), treated with the indicated concentrations of ethanol and/or atorvastatin. The error bars represent the SD from the mean of five assays of an individual experiment. *Significant difference, ethanol treatment versus untreated control (1 way ANOVA with multiple comparisons; n = 5; *P < 0.05, **P < 0.01, ***P < 0.001 vs. control)

Fig. 6

Major metabolic pathways of ethanol in humans. (Adapted from [23, 36, 38]). Orally ingested alcohol (EtOH) is rapidly oxidized in the liver to acetaldehyde, primarily by alcohol dehydrogenase(s), and secondarily by the microsomal ethanol oxidizing system (MEOS, including cytochrome P450 CYP2E1) or by peroxisomal catalase. Acetaldehyde is further rapidly oxidized to acetate, which is either converted directly to acetyl-CoA in the liver, or released to plasma, from where it is ultimately metabolized primarily to CO₂ in peripheral tissues. Most ethanolic carbon is ultimately released as CO₂. A small fraction of ethanolic carbon in acetyl-CoA is converted to free fatty acids (FFA) or cholesterol (cholest) via de novo lipid synthesis. A small fraction of ingested ethanol may be non-oxidatively metabolized to fatty acid ethyl ester (FAAE) or phosphatidyl ethanol (PTE). Acetyl-CoA synthetase 2 (ACSS2) also transfers acetate to chromosomal histones [39]. Octagons around EtOH and FFA/cholest denote that inhibitor studies indicate that these are not candidates for primary signaling molecules for ApoA1 upregulation