Molecular and Cellular Pharmacology
Regulation mechanism of ABCA1 expression by statins in hepatocytes

https://doi.org/10.1016/j.ejphar.2011.04.043Get rights and content

Abstract

ATP-binding cassette transporter A1 (ABCA1) is predicted to be involved in the control of apolipoprotein AI-mediated cholesterol efflux: biosynthesis of high-density lipoprotein (HDL). However, the effects of HMG-CoA reductase inhibitors (statins) on ABCA1 in the liver and the precise mechanisms of their actions have been obscure. The aims of this study were to determine whether statins (atorvastatin (Ato) and pitavastatin (Pit)) affect hepatic ABCA1 expression and to clarify the mechanisms of their actions using HepG2 cells and the rat liver. We examined alterations in mRNA and protein levels of ABCA1 and peroxisome proliferator-activated receptors (PPARs) by quantitative real-time polymerase chain reaction (PCR) and Western blot analysis, respectively. In vitro and in vivo studies suggested that Pit increases ABCA1 mRNA level, but not Ato. Pit greatly increased Abca1 mRNA level and also increased the amount of plasma HDL and the mRNA level of PPARα. Clofibrate (PPARα agonist) increased ABCA1 expression in HepG2 cells and rat primary hepatocytes more than did PPAR β/δ and γ agonists. Pit-induced ABCA1 expression alteration was blocked by GW6471 (PPARα antagonist) and by PPARα knockdown. In this study, we demonstrated that Pit affect ABCA1 expression via PPARα in hepatocytes. The strategy to target a PPARα agonist in the liver can lead to increases in ABCA1 expression and HDL level.

Introduction

HMG-CoA reductase inhibitors (statins) reduce low-density lipoprotein (LDL) cholesterol concentration through blockade of the mevalonate pathway and consequent increment of LDL receptor expression in the liver (Goldstein and Brown, 1990). Statins are the most widely used cholesterol-lowering agents for prevention of cardiovascular disease (Havel and Rapaport, 1995). Atherosclerotic diseases including cardiovascular disease are the main cause of death in individuals with metabolic syndrome. The reverse cholesterol transport system including ATP-binding cassette transporter A1 (ABCA1) and high-density lipoprotein (HDL) plays an important role in atherosclerosis (Chinetti et al., 2006). ABCA1 expression in liver was shown to be necessary for HDL formation. Inactivation of ABCA1 gene in mice leads to a severe HDL deficiency (Aiello et al., 2003), and targeted disruption of ABCA1 gene in mouse hepatocytes reduces plasma HDL level by 80% (Lee and Parks, 2005). On the other hand, overexpression of ABCA1 in mouse liver markedly increases plasma HDL level (Wellington et al., 2003). However, the effects of statins on ABCA1 in the liver and the precise mechanisms of their actions have been obscure.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily. Three subtypes of PPARs, PPARα, PPARβ/δ and PPARγ, have been identified. PPARα is predominantly expressed in the liver, kidney, heart and skeletal muscle, where it controls fatty acid metabolism. PPARβ/δ is ubiquitously expressed and controls brain lipid metabolism and fatty acid-induced adipogenesis and preadipocyte proliferation. PPARγ, which is highly expressed in brown and white adipose tissue and the intestine, triggers cellular differentiation, promotes lipid storage and modulates the action of insulin (Torra et al., 2001). ABCA1 gene expression is known to be regulated by PPARα (Ogata et al., 2009) and PPARγ (Chawla et al., 2001). Although recent studies have demonstrated that PPARα mRNA level was affected by statins in an in vitro study (Seo et al., 2008), it has remained unclear that PPARα or the other isoforms, PPARβ/δ and PPARγ were affected by statins in vitro and in vivo.

The aims of this study were to determine whether statins (atorvastatin (Ato) and pitavastatin (Pit)) affect hepatic ABCA1 expression and to clarify the mechanisms of their actions.

Section snippets

Chemicals

Ato was kindly donated by Sankyo (Tokyo, Japan). Pit was kindly donated by Kowa (Tokyo, Japan). Clofibrate and GW6471 were obtained from Sigma-Aldrich (St. Louis, MO). All other reagents were of the highest grade available and used without further purification.

Cell culture

HepG2 cells were kept in Dulbecco's modified Eagle's medium (Sigma Aldrich Japan, Tokyo) with 10% fetal bovine serum (ICN Biomedicals, Inc., Aurora, OH) and 1% penicillin-streptomycin at 37 °C under 5% CO2 as described previously (

Effects of statins on ABCA1 expression in hepatocytes

First, we examined the alteration of ABCA1 mRNA level induced by Ato and Pit in HepG2 cells. Pit significantly up-regulated ABCA1 mRNA level. On the other hand, Ato had little effect (Fig. 1A). To investigate whether Pit regulated ABCA1 mRNA level in vivo, Pit were given to rats. Pit significantly increased Abca1 mRNA level in the rat liver, but not Ato (Fig. 1B). The results of in vitro and in vivo studies suggest that Pit increase ABCA1 expression in hepatocytes and that the effects of

Discussion

In the present study, we found that Pit induced ABCA1 expression in both HepG2 cells and the rat liver. As tested in HepG2 cells, this inductive effect of Pit was mediated through a PPARα-dependent pathway. In an in vitro study, Pit increased mRNA level of ABCA1, but Ato had little effect on the mRNA level (Fig. 1). Moreover, Pit significantly increased plasma HDL concentration. On the other hand, Ato had no effect (Table 2). Yokote et al. (2008) reported that a significant increase in HDL-C

References (22)

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1

These authors contributed equally as first author.

2

Present address: Department of Pharmacy, Hirosaki University School of Medicine and Hospital, Hirosaki 036-0563, Japan.

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