Pravastatin Modulate Niemann-Pick C1-Like 1 and ATP-Binding Cassette G5 and G8 to Influence Intestinal Cholesterol Absorption

- Purpose. Niemann-Pick C1-like 1 (NPC1L1), ATP-binding cassette (ABC)G5, and ABCG8 mediate intestinal cholesterol absorption. It is unclear whether pravastatin (PR) or ezetimibe (EZ) affect expression of these transporters. We examined the effects of PR and EZ on NPC1L1, ABCG5, and ABCG8 expression in human hepatoma HepG2 cells and the murine small intestine. We also assessed expression of the transcription factors liver X receptor (LXR)  , LXR  and sterol regulatory element-binding protein. Methods. Transporter mRNA levels were determined in murine small intestines 6 and 24 h after oral PR and EZ administration by real-time reverse-transcriptase polymerase chain reaction (RT-PCR). In PR- and EZ-treated HepG2 cells, transporter and transcription factor mRNA and protein levels were examined by RT-PCR and western blot, respectively. Results. Significant decreases in NPC1L1 , ABCG5 , and ABCG8 mRNA expression were observed in the duodenum, but not jejunum and ileum, of mice 24 h after treatment with PR, but not EZ. In HepG2 cells, PR but not EZ treatment for 24 h also significantly decreased NPC1L1 protein and ABCG5 , and ABCG8 mRNA expression, while increasing LXR  mRNA levels. Conclusion. PR but not EZ treatment reduced duodenal cholesterol transporter expression in mice. PR-induced increases in LXR  mRNA levels may be involved in attenuation of NPC1L1 expression, subsequently


INTRODUCTION
Cholesterol homeostasis in the body is maintained by biosynthesis, intestinal absorption, and biliary excretion. Niemann-Pick C1-like 1 (NPC1L1) is expressed in the epithelial cells of the duodenum, jejunum, and proximal ileum, where it mediates intestinal cholesterol and phytosterol absorption (1)(2)(3). Statins, such as pravastatin (PR), suppress 3hydroxy-3-methylglutaryl coenzyme A reductase activity, and regulate hepatic low-density lipoprotein receptors in animals and humans (4). Ezetimibe (EZ) inhibits intestinal cholesterol absorption via NPC1L1 to reduce plasma cholesterol levels (5)(6)(7). Thus, NPC1L1 is a therapeutically important molecular target for dyslipidemia, metabolic syndromes, and obesity. Additionally, ATP-binding cassette (ABC)G5 and ABCG8, which are located on the apical membrane of enterocytes, and are regulated by the liver X receptor (LXR) (8,9), play important roles in cholesterol homeostasis by mediating its efflux (10,11). Genetic ablation of ABCG5 and ABCG8 in mice resulted in disruption of cholesterol homeostasis (12).
When combined, PR and EZ caused a synergistic reduction in plasma cholesterol levels in patients with dyslipidemia (13). However, it is unclear whether PR and EZ affect the expression of transporters involved in cholesterol absorption, such as NPC1L1, ABCG5, and ABCG8. As plasma cholesterol levels could be affected by the intestinal expression and activity of NPC1L1, ABCG5, and ABCG8, it is important to elucidate the effects of PR and EZ on these proteins.
To investigate the effects of PR and EZ on NPC1L1, ABCG5, and ABCG8, we examined the expression levels of these genes in human hepatoma HepG2 cells, and the murine small intestine, following PR and EZ treatment. HepG2 cells express NPC1L1, ABCG5, and ABCG8 and are used for functional evaluation of these genes (14,15). We also determined mRNA levels of the transcription factors LXR, LXR and sterol regulatory elementbinding protein (SREBP)2 in HepG2 cells. _________________________________________

Compounds and reagents
PR (sodium salt) and EZ were purchased from LKT Laboratories (St Paul, MN, USA) and Sequoia Research Products (Pangbourne, UK), respectively. GlutaMAX supplement I and TRIzol were obtained from Life Technologies (Carlsbad, CA, USA). All other chemicals and solvents were of the highest purity commercially available.

Animals and treatment
Five-week-old male C57BL/6 mice were purchased from Japan SLC (Shizuoka, Japan). Animals were housed in a temperature-controlled room with free access to standard laboratory rodent food (MF diet, Oriental Yeast, Tokyo, Japan) and water. Mice were treated orally once with PR (5 mg/kg or 15 mg/kg) and EZ (5 mg/kg). We chose the dose of PR and EZ for previous pharmacokinetics studies (16)(17)(18). The plasma PR concentrations could be comparable to clinical therapeutic concentrations (19). After administering anesthesia by diethyl ether inhalation and sacrifice by cervical dislocation, the duodenum (1-8 cm from the stomach), jejunum (10-18 cm from the stomach), and ileum (8 cm above the cecum) were excised 6 (short-term) and 24 h after PR or EZ injection. Each sample was flash frozen in liquid nitrogen and preserved at −80°C until RNA extraction. The study protocol was approved by the Committee for the Care and Use of Laboratory Animals of the School of Pharmacy of Kinki University (Osaka, Japan).

Determination of mRNA levels by real-time reverse transcription polymerase chain reaction (RT-PCR)
Total RNA was extracted from murine small intestines and HepG2 cells using TRIzol. mRNA expression was measured using RT-PCR, as described previously (20,21). The oligonucleotide sequences for each mRNA target are shown in Table  1. Data were analyzed using ABI Prism 7000 SDS software (Life Technologies), using the multiplex comparative method.

STATISTICAL ANALYSES
Significant differences between mean values were determined by analysis of variance followed by the Bonferroni correction. Significance levels were determined at p < 0.05.

RESULTS
Relative NPC1L1, ABCG5, and ABCG8 mRNA levels in the duodenum, jejunum, and ileum of mice orally administered PR (5 mg/kg and 15 mg/kg) at 24 h are shown in Fig. 1. Following PR treatment, NPC1L1, ABCG5, and ABCG8 mRNA levels in the duodenum were significantly lower, with dramatic decreases at higher PR doses (15 mg/kg). However, in the jejunum and ileum, few changes in NPC1L1, ABCG5, and ABCG8 mRNA levels were observed following PR treatment. To clarify whether expression of these transporters were affected by 6 hr (short-term) after PR administration, NPC1L1, ABCG5, and ABCG8 mRNA levels in the duodenum were also determined 6 h post-treatment; however, no expression changes were observed at this time point (Fig. 2). NPC1L1, ABCG5, and ABCG8 mRNA expression in the duodenum of mice treated orally with EZ (5 mg/kg) at 24 h are shown in Fig.  2; mRNA levels of these genes were not altered by EZ treatments.  We also examined effects of PR and EZ on NPC1L1, ABCG5, and ABCG8 expression in vitro using HepG2 cells. Dose-and time-dependent effects of these compounds on NPC1L1, ABCG5, and ABCG8 mRNA levels are shown in Fig. 3a and 3b. NPC1L1, ABCG5, and ABCG8 mRNA expression were dose-dependently suppressed by PR treatments (Fig. 3a). At 30 and 300 M PR, NPC1L1 and ABCG8 mRNA levels were significantly decreased in HepG2 cells. When we analyzed the time course of NPC1L1 mRNA levels following PR treatment, NPC1L1 expression was significantly reduced after 24 h (Fig. 3b). We also assessed the effects of PR on NPC1L1 protein levels (Fig. 3c). NPC1L1 mRNA and protein levels showed corresponding significant decreases following PR administration (at 30 and 300 M). Similar NPC1L1, ABCG5, and ABCG8 mRNA levels to baseline controls were observed at each EZ-treatment concentration in HepG2 cells (Fig. 4).
To investigate the underlying mechanisms of PR-induced changes in NPC1L1, ABCG5, and ABCG8 levels, we examined mRNA expression of the transcription factors LXR, LXR and SREBP2 (Fig. 5). Significant increases in LXR mRNA levels were observed following treatment with 3-300 M PR. Higher LXR mRNA levels were also observed following administration of 30 M PR; however, few changes in SREBP2 expression were observed.

DISCUSSION
Intestinal cholesterol absorption via NPC1L1 and its de novo synthesis are important factors for cholesterol homeostasis. Because PR and EZ affect expression of the intestinal cholesterol-absorbing proteins NPC1L1, ABCG5, and ABCG8, the present study demonstrates that these compounds are potentially therapeutic drugs for dyslipidemia. NPC1L1, ABCG5, and ABCG8 mRNA levels were significantly decreased in the duodenum, but not jejunum and ileum, of mice 24 h after PR treatment (Fig. 1), indicating that PR regionally affected the expression of these genes in the small intestine of mice. NPC1L1 is highly expressed in the duodenum and jejunum, but not other tissues, in mice (1,3,22). PR reaches a maximum plasma concentration within 2 h of oral administration, and is eliminated with a relatively short half-life (1-3 h; 23, 24). PR was primarily absorbed from the duodenum (25). Thus, the region-specific effects of PR may have been the result of higher duodenal concentrations (compared with those in the jejunum and ileum) after oral administration.
Although PR and EZ both pharmacologically act to reduce plasma cholesterol levels (26), NPC1L1, ABCG5, and ABCG8 mRNA expression did not change following EZ treatment (Figs. 2 and 4).   Unfortunately, the precise mechanism underlying the differential effects of PR and EZ on the expression of these genes is unclear. The primary sites of PR and EZ action are the small intestine and liver, respectively, which could partially account for the observed differential effects on NPC1L1, ABCG5, and ABCG8 expression. Further studies are needed to clarify whether higher doses or repeated administration of EZ affect the expression of these genes.
In our in vitro experiments using HepG2 cells, the examined PR and EZ concentrations did not promote lactate dehydrogenase leakage (data not shown), suggesting that cytotoxicities induced by PR and EZ were minimal. In accordance with the results of our in vivo experiments, PR, but not EZ, treatment suppressed NPC1L1, ABCG5, and ABCG8 mRNA expression in HepG2 cells. LXR mRNA was also significantly increased in HepG2 cells treated with PR (Fig. 5). However, the effects of PR on LXR protein levels and activity remain unclear. LXR and LXR belong to the nuclear receptor family, and are crucial inhibitory NPC1L1 regulators (27)(28)(29). Thus, the present results suggest that PRinduced LXRupregulation may attenuate duodenal NPC1L1 expression.
It has been reported that cholesterol intake upregulates ABCG5 and ABCG8 expression in the small intestine (30). Moreover, cholesterol-free diet has been found to suppress ABCG5 and ABCG8 gene expression in the murine small intestine (31). Thus, because PR inhibits cholesterol synthesis in the small intestine and liver, PR may also attenuate ABCG5 and ABCG8 expression by such a mechanism.
We determined the effects of single PR and EZ administrations on NPC1L1, ABCG5, and ABCG8 the mRNA levels in the small intestine. However, in clinical settings, PR and EZ used long term. Previous reports showed that 6 weeks of statin therapy was associated with an increase in intestinal cholesterol absorption (32). Moreover, atorvastatin decreased cholesterol synthesis, while increasing intestinal NPC1L1 expression in dyslipidemic men (33), suggesting that reducing cholesterol levels increases the expression of this gene. We have previously shown that NPC1L1 expression was significantly decreased in the small intestine of mice fed a highcholesterol diet for 7 days (34). These long-term results differed from those obtained using a single PR administration in this study. Thus, NPC1L1 feedback regulation in long-term statin treatment should be evaluated separately from that of single administrations. Further studies are needed to clarify the effects of repeated PR and EZ administration.
The effects of other nutrients on NPC1L1 expression have also been reported. For example, Dglucose directly modulated NPC1L1 expression via transcriptional mechanisms and phosphatasedependent pathways (35). Additionally, dietarycalcium-induced plasma total cholesterol reductions were accompanied by transcriptional NPC1L1 downregulation, along with ABCG5 and ABCG8 upregulation (36). Whether other drugs or nutrients affect intestinal NPC1L1, ABCG5, and ABCG8 expression or cholesterol absorption remains to be investigated.

CONCLUSION
In conclusion, we demonstrated that treatment with PR, but not EZ, decreased NPC1L1 expression in the duodenum, tentatively as the result of LXR upregulation. Moreover, our results show that a single administration of PR could affect intestinal cholesterol absorption.