Pravastatin activates PPARα/PPARγ expression in the liver and gallbladder epithelium of hamsters

Seoul, Korea

Pravastatin activates PPARα/PPARγ expression in the liver and gallbladder epithelium of hamsters

Seok Ho Dong, Jin Lee, Dong Hee Koh, Min Ho Choi, Hyun Joo Jang and Sea Hyub Kae

Seoul, Korea

BACKGROUND: Our earlier study with cultured gallbladder epithelial cells demonstrated that statins (HMG-CoA reductase inhibitors) activate the expression of PPARα and PPARγ, consequently blocking the production of pro-inflmmatory cytokines. The present study used hamsters to investigate the effects of pavastatin on PPARα/PPARγ expression in the liver and gallbladder epithelium, and to determine whether pravastatin suppresses cholesterol crystal formation in the gallbladder.

METHODS: A total of 40 Golden Syrian male hamsters (4 weeks old) were randomly assigned to four groups (basal diet control; basal diet+pavastatin; high cholesterol diet; high cholesterol diet+pravastatin). All hamsters were 11 weeks old at the end of the experiment. The liver, gallbladder and bile were harvested. Immunohistochemical staining and Western blotting for PPARα and PPARγ were performed in the liver and gallbladder. A drop of fresh bile was examined for cholesterol crystals under a microscope.

RESULTS: In the gallbladder and liver of the hamsters, pravastatin activated the PPARα and PPARγ expression of gallbladder epithelial cells and hepatocytes, and particularly the response of PPARγ was much stronger than that of PPARα. Pravastatin suppressed the formation of cholesterol gallstones or crystals in the gallbladder.

CONCLUSION: Pravastatin is an effective medication to activate PPARs (especially PPARγ) in the liver and the gallbladder epithelium of hamsters, and contributes to the prevention of gallstone formation.

(Hepatobiliary Pancreat Dis Int 2011; 10: 185-190)

pravastatin; PPARα; PPARγ; hamster; gallstone

Introduction

Gallstones are the leading cause of gastrointestinal morbidity in most countries, sometimes presenting as serious complications such as acute necrotizing pancreatitis or suppurative cholangitis.[1-3]Currently the standard strategy for treatment of gallstones is laparoscopic cholecystectomy when a symptom or a complication develops.[4]However, it may also be important to prevent gallstone formation in patients with high risk factors such as hyperlipidemia, immobility, obesity, and old age who have serious underlying diseases.

Our earlier studies with cultured gallbladder (GB) epithelial cells demonstrated that PPARα and PPARγ ligands modulate inflammation by suppressing TNF-α production and preventing excessive accumulation of cholesterol by ABCA1 activation.[5]We also documented that statins (HMG-CoA reductase inhibitors) activate PPARα and PPARγ expression, consequently also blocking the production of pro-inflammatory cytokines induced by lipopolysaccharide. We postulated that statins can be a safe and effective medication to preserve GB function and to prevent gallstone formation.[6]

Several clinical studies[7,8]have been performed to determine whether statins can dissolve cholesterol gallstones or crystals. However, the results of these studies are inconsistent. Animal studies also showed considerable disagreement regarding the preventive effects of statins on gallstone or crystal formation.[9-11]Recently it has been reported that long-term use of statins is associated with a decreased risk of gallstones followed by cholecystectomy.[12]

However, so far no animal studies have been performed to evaluate the effects of statins on PPARα/ PPARγ expression in the liver and GB where bile is produced. Therefore, we designed this animal studyusing hamsters to evaluate the effects of pravastatin on PPARα/PPARγ expression in the liver and GB epithelium, and to assess the preventive effect of pravastatin on gallstone or crystal formation in the GB.

Methods

Animals, diet and sampling of bile and organs

Four-week-old male Golden Syrian hamsters (Mesocricetus auratus) weighing 50-60 g were purchased from Biolink Korea (Seoul, Korea). Forty hamsters were randomly assigned to four groups (BD, basal diet; BDP, basal diet+pravastatin; CD, high cholesterol diet; and CDP, high cholesterol diet+pravastatin). The hamsters were housed in groups of 5 in a plastic cage containing wood shavings and maintained in a room with alternating 12-hour periods of light and darkness. They were adapted to these conditions for 1 week, during which they were fed a pelleted form of a cereal-based rodent diet (Feedlab, Seoul, Korea), defined as the basal diet. This diet had a cholesterol content of 0.03% (wt/ wt) and a total lipid content of 4%-5% (wt/wt). In the high cholesterol diet group, the meal form of the basal diet was enriched with cholesterol (0.3% wt/wt). The total amount of feed was restricted to 12 g/day for each hamster, based on the usual daily consumption. In the studies with the pravastatin group, pravastatin (Hanmi Pharm, Seoul, Korea) was provided in the diet at a single dose of 10 mg/kg body weight per day. Body weight and feed consumption were monitored throughout the experiments.

All hamsters were 11 weeks old at the end of the experiment. The animals were anesthetized by intraperitoneal injection of xylazine (Rompun?; 5 mg/kg body weight) and zolazepam (Zoletil?; 50 mg/kg body weight). The abdomen was opened with a mid-line incision, and the GB was examined for the presence of gallstones. The bile was collected with a 50 μl Hamilton syringe and stored at -70 ℃ until use. A fresh drop of bile was observed under a light microscope to confirm the presence of crystals. The GB was removed, opened, rinsed with saline and stored in liquid nitrogen for subsequent tissue analysis. The liver was rapidly excised and stored in liquid nitrogen for subsequent tissue analysis. The hamsters were finally sacrificed by cervical dislocation. The care and use of the animals for experimental purposes were in accordance with the NIH Guide for the Care and Use of Laboratory Animals.[13]

Immunohistochemical assay

The excised GB and liver tissues were fixed in 4% paraformaldehyde for 24 hours. After fixation, the tissue block was embedded in paraffin, then cut on a microtome into 4 μm sections and affixed to slides. The sections were stained immunohistochemically using a diaminobenzine peroxidase-antiperoxidase technique. The slides were deparaffinized and blocked for 5 minutes with 3% hydrogen peroxide to block endogenous peroxidase activity. Antigen activation was performed by microwave for 10 minutes; then the slides were immersed in boiled Dako Epitope Retrieval solution (0.01 mol/L citrate buffer, pH 6.0) (Dako, Carpinteria, CA, USA). The sections were incubated with rabbit polyclonal PPARα or rabbit polyclonal PPARγ (Abcam, Cambridge, UK) for 40 minutes at room temperature. For incubation, primary antibodies were diluted in 1∶200 in antibody diluent (Dako, Carpinteria, CA, USA). After washing in PBS, the sections were incubated with horseradish peroxidaseconjugated goat anti-rabbit IgG (Dako) for 30 minutes at room temperature. The complex was visualized using 3, 3'-diaminobenzidine tetrahydrochloride (Dako) for 5 minutes after washing in PBS. The sections were counterstained with hematoxylin and observed under a light microscope. Negative control was done by omitting the primary antibody. The nuclear and cytoplasmic staining results of PPAR were graded as 0 (no staining), 1 (weak staining), 2 (moderate staining), or 3 (strong staining) by comparison with the negative control.

Protein extraction from tissues and Western blotting

The frozen liver tissues were homogenized with a mechanical homogenizer for 30 minutes at 4 ℃followed by incubation in lysis buffer (50 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 μmol/L phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 5 μg/ml leupeptin). The supernatant was harvested after centrifugation at 12 000 rpm for 20 minutes at 4 ℃. Extracted total protein was quantified by the Bradford assay using bovine serum albumin (Sigma, St Louis, MO, USA). SDS-PAGE was performed with a 4% stacking gel and an 8% resolving gel, followed by transfer to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked for 1 hour at room temperature in blocking solution (5% skim milk in Tris-buffer with Tween-20 (TBS-T): 20 mmol/L Tris, 500 mmol/L NaCl, pH 7.5, 0.05% v/v Tween-20). They were then incubated with rabbit polyclonal PPARα or rabbit polyclonal PPARγ (Santa Cruz Biotechnology, CA, USA) overnight at 4 ℃. At the same time, the rabbit polyclonal β-actin antibody was used as a housekeeping protein to normalize the amount of protein. The membranes were washed with TBS-T and incubatedwith peroxidase-conjugated anti-rabbit IgG (Amersham, Buckinghamshire, UK) for 1 hour at room temperature. The membrane was washed and incubated using an ECL Western blot detection kit (Amersham) for 5 minutes. Next, autoradiography was performed. The signal intensities of specific bands on Western blotting were quantified using NIH Image J density analysis software (version 1.20).

Statistical analysis

All results described are representative of at least three separate experiments. Results from each experiment are expressed as the mean±SD of duplicate assays. To analyze the Western blotting assays, one-way analysis of variance (ANOVA) for three or more unpaired groups or Student'sttest for two unpaired groups was used. To analyze the immunohistochemical assays, the Mann-WhitneyUtest or the Kruskal-Wallis test was used. Fisher's exact test was used to analyze the experiment on stones or crystals in the GB. APvalue less than 0.05 was considered statistically significant.

Results

Pravastatin activated PPARα and PPARγ expression in GB epithelial cells

Fig. 1. Immunohistochemical analysis of PPARα in the GB (original magnification ×400). In the BDP (B) or CD (C) group, only cytoplasmic PPARα expression in GB epithelial cells was increased compared to the BD group (A) or negative control, but not in the nuclei. In the CDP (D) group, PPARα expression in the nuclei of GB epithelial cells was increased compared to the BD (control), BDP, and CD groups (P＜0.001). BD: basal diet; CD: high cholesterol diet; BDP: basal diet with pravastatin; CDP: high cholesterol diet with pravastatin.

Immunohistochemical staining was performed to investigate the effects of pravastatin on PPARα and PPARγ expression in GB epithelial cells. In the BDP and CD groups, only cytoplasmic PPARα expression was increased compared to the BD group and negative control, but not in the nuclei. In the CDP group, PPARα expression in the nuclei was increased compared to the BD, BDP, and CD groups (P＜0.001) (Fig. 1). In case of PPARγ expression, stronger activation was induced in the BDP, CD, and CDP groups than in the BD group (P＜0.001) (Fig. 2), which also showed that pravastatin and cholesterol feeding activated PPARγ expression more strongly than PPARα.

Pravastatin activated PPARα and PPARγ expression in hepatocytes

In immunohistochemical assays, the BDP (P＜0.01, vs. BD), CD (P＜0.01, vs. BD), and CDP groups (P＜0.001, vs. BD;P＜0.01 vs. all other groups) showed increased expression of PPARα compared to the BD group (Fig. 3). The BDP (P＜0.001, vs. BD), CD (P＜0.001, vs. BD), and CDP groups (P＜0.001, vs. BD;P＜0.01, vs. BDP) also showed increased expression of PPARγ compared to the BD group (Fig. 4).

In Western blotting assays (Fig. 5), addition of pravastatin to the diet (BDP and CDP groups) activated the expression of PPARγ (P＜0.001, vs. groups without pravastatin). PPARγ expression was stronger in the CD group than in the BD group (P＜0.001), and was stronger in the BDP group than in the CD group (P＜0.001). The strongest expression of PPARγ occurred in the CDPgroup (P＜0.001, vs. all other groups). In case of PPARα, the CDP and CD groups showed increased expression (P＜0.001) compared to the BD group, but not as strong as PPARγ .

Fig. 2. Immunohistochemical analysis of PPARγ in the GB (original magnification ×400). Stronger activation was induced in the BDP (B), CD (C), and CDP (D) groups than in the BD (A) group (control) (P＜0.001), which also showed that pravastatin and cholesterol activated PPARγ expression more strongly than PPARα. BD: basal diet; CD: high cholesterol diet; BDP: basal diet with pravastatin; CDP: high cholesterol diet with pravastatin.

Fig. 3. Immunohistochemical analysis of PPARα in the liver (original magnification ×400). Expression of PPARα in the BDP (B), CD (C), and CDP groups (D) significantly increased compared to the BD group (A) (control). BD: basal diet; CD: high cholesterol diet; BDP: basal diet with pravastatin; CDP: high cholesterol diet with pravastatin.

Fig. 4. Immunohistochemical analysis of PPARα in the liver (original magnification ×400). Expression of PPARγ in the BDP (B), CD (C), and CDP groups (D) significantly increased compared to the BD group (A) (control). BD: basal diet; CD: high cholesterol diet; BDP: basal diet with pravastatin; CDP: high cholesterol diet with pravastatin.

Pravastatin suppressed formation of cholesterol gallstones or crystals

Table. Body weight change during experimental period and incidence of gallstone or crystal formation in GB at the end of experiment

Body weight after 6 weeks and feed consumption during experimental period were not significantly different among the four groups (Table). In order to demonstrate the preventive effects of pravastatin on gallstones in the GB, a fresh drop of bile was observed under a light microscope to confirm the presence of crystals, or the opened GB was grossly observed. No gallstones or crystals were found in bile from the BD and BDP groups. However, gallstones or crystals were found in 8/10 of the CD group, and 3/9 of the CDP group (P＜0.001 vs. CD) (Table). Pravastatin, thus, significantly suppressed the formation of cholesterol gallstones or crystals induced by the high cholesterol diet.

Discussion

Despite some disagreement with regard to the preventive or dissolving effects of statins on gallstones or crystals, most of the clinical and animal studies consistently suggest that statins reduce the cholesterol in bile.[7-10]In addition, a well-designed animal study with prairie dogs has shown that lovastatin treatment reduces bile cholesterol, altering bile acid composition, and inducinga higher dissolution of stones compared to placebo.[11]Until recently, the long-term effect of statins in preventing gallstone formation has remained unresolved, because most studies were performed in a short period. However, a large-scale case-control analysis finally revealed that long-term use of statins over one year is associated with a decreased risk of gallstones followed by cholecystectomy.[12]With our result that pravastatin suppressed the formation of cholesterol crystals and the data from recent studies, we suggest that statins can lower cholesterol concentration in bile and prevent gallstone or crystal formation. Accordingly, statins may provide a choice to prevent gallstone formation in patients with high risk factors such as hyperlipidemia, immobility, obesity, and old age who have serious underlying diseases.

Mechanisms involved in the preventive effect of statins on gallstone formation are not fully understood yet. The following are the possible hypotheses. First, statins decrease hepatic cholesterol biosynthesis and may thereby decrease biliary cholesterol secretion, consequently leading to diminished cholesterol concentration in bile.[14]Second, statins activate PPARs (especially PPARγ) that are associated with the suppression of gallstone formation.[6]A study reported that the generation failure of endogenous PPARγ agonists leads to cholesterol supersaturation in bile and gallstone formation,[15]and another study suggested that decreased hepatic expression of PPARγ coactivator-1 is associated with the presence of cholesterol gallstones.[16]In this animal study, we demonstrated that pravastatin activated the expression of PPARs in the liver and GB epithelium, similar to the results of the study performed with cultured GB epithelial cells.[6]On the other hand, PPARγ agonists have never been associated with the risk of gallstone formation, and the current study showed that expressional change of PPARγ was more prominent than PPARα in hepatocytes after feeding with statin. This finding may explain why statins do not increase the cholesterol saturation index (CSI), despite the facts that PPARα ligands (fibrates) increase the risk of cholesterol gallstone formation and that statins activate PPARα.[17]

Early, we demonstrated that statins have a powerful anti-inflammatory effect by blocking lipopolysaccharide-induced TNF-α production in cultured GB epithelial cells,[6]which is consistent with our previous results that PPAR ligands suppress lipopolysaccharideinduced TNF-α production in cultured GB epithelial cells.[5]Statins are involved in the anti-inflammatory process in GB epithelial cells, not only by blocking the activity of NF-κB directly but also activating mainly PPARγ that can abolish NF-κB activity.[18]We have also suggested that statin induces activation of ABCA1 expression via the LXRα-mediated pathway that can play a role in eliminating excessively loaded cholesterol in GB epithelial cells, preventing chronic inflammation and maintaining motility in the GB.[19,20]In this study, the activation of PPARs was found in both GB epithelial cells and hepatocytes after high cholesterol feeding. We speculate that PPARs are activated to suppress inflammation as a compensatory response to oxidized cholesterol in the liver and GB epithelium.

The lipophilicity of statins is important because their hepatoselectivity is associated with lipophilicity. More lipophilic statins have a tendency to reach higher levels of exposure in non-hepatic tissues while hydrophilic statins tend to be more hepatoselective. The difference in selectivity is because lipophilic statins passively and non-selectively diffuse into both hepatocytes and nonhepatocytes, while hydrophilic statins depend largely on active transport into hepatocytes.[21,22]It is known that high hepatoselectivity is considered to translate into reduced risk of adverse effects.[23]The major target organs of our experiments were the liver and GB, and reduction of the toxicity of statin during feeding was thought to be important. Accordingly, we chose hydrophilic pravastatin for this study.

A limitation of this study is that bile composition analysis for calculation of CSI was not performed. The amount of bile harvested from each hamster was not enough for analysis due to technical difficulties. However, because the primary objectives of the study were to demonstrate changes in the expression of PPARs and to observe crystals or stones in bile, this limitation did not affect the conclusions that pravastatin is an effective medication to activate PPARs in the liver and GB epithelium, and contributes to the prevention of gallstone formation. In addition, another study from our group (ready for submission) regarding the genes associated with CSI in bile demonstrates that pravastain activates the expression of FXR and 7α-hydroxylase mRNA as well as LXRα-mediated ABCG5/ABCG8 mRNA, and these results indicate that pravastain decreases or does not increase CSI in bile. In future, we anticipate studies with long-term and prospective clinical trials to reveal the actual effects of statins on the prevention of gallstone formation in the GB.

Funding: This study was supported by a grant from Kyung Hee University Research (20071618).

Ethical approval: Not needed.

Contributors: DSH and LJ proposed the study. LJ wrote the first draft. DSH and LJ analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. LJ is the guarantor.Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

1 Shaffer EA. Epidemiology and risk factors for gallstone disease: has the paradigm changed in the 21st century? Curr Gastroenterol Rep 2005;7:132-140.

2 Russo MW, Wei JT, Thiny MT, Gangarosa LM, Brown A, Ringel Y, et al. Digestive and liver diseases statistics, 2004. Gastroenterology 2004;126:1448-1453.

3 Everhart JE, Khare M, Hill M, Maurer KR. Prevalence and ethnic differences in gallbladder disease in the United States. Gastroenterology 1999;117:632-639.

4 Keus F, Broeders IA, van Laarhoven CJ. Gallstone disease: Surgical aspects of symptomatic cholecystolithiasis and acute cholecystitis. Best Pract Res Clin Gastroenterol 2006;20:1031-1051.

5 Lee J, Hong EM, Byun HW, Choi MH, Jang HJ, Eun CS, et al. The effect of PPARalpha and PPARgamma ligands on inflammation and ABCA1 expression in cultured gallbladder epithelial cells. Dig Dis Sci 2008;53:1707-1715.

6 Lee J, Hong EM, Koh DH, Choi MH, Jang HJ, Kae SH, et al. HMG-CoA reductase inhibitors (statins) activate expression of PPARalpha/PPARgamma and ABCA1 in cultured gallbladder epithelial cells. Dig Dis Sci 2010;55:292-299.

7 Smit JW, van Erpecum KJ, Renooij W, Stolk MF, Edgar P, Doornewaard H, et al. The effects of the 3-hydroxy, 3-methylglutaryl coenzyme A reductase inhibitor pravastatin on bile composition and nucleation of cholesterol crystals in cholesterol gallstone disease. Hepatology 1995;21:1523-1529.

8 Smit JW, van Erpecum KJ, Stolk MF, Geerdink RA, Cluysenaer OJ, Erkelens DW, et al. Successful dissolution of cholesterol gallstone during treatment with pravastatin. Gastroenterology 1992;103:1068-1070.

9 Tazuma S, Hatsushika S, Aihara N, Sagawa H, Yamashita G, Sasaki M, et al. Inhibitory effects of pravastatin, a competitive inhibitor of hydroxymethylglutaryl coenzyme A reductase, on cholesterol gallstone formation in prairie dogs. Digestion 1992;51:179-184.

10 Davis KG, Wertin TM, Schriver JP. The use of simvastatin for the prevention of gallstones in the lithogenic prairie dog model. Obes Surg 2003;13:865-868.

11 Abedin MZ, Narins SC, Park EH, Smith PR, Kirkwood KS. Lovastatin alters biliary lipid composition and dissolves gallstones: a long-term study in prairie dogs. Dig Dis Sci 2002;47:2192-2210.

12 Bodmer M, Brauchli YB, Kr?henbühl S, Jick SS, Meier CR. Statin use and risk of gallstone disease followed by cholecystectomy. JAMA 2009;302:2001-2007.

13 McPherson C. Regulation of animal care and research? NIH's opinion. J Anim Sci 1980;51:492-496.

14 Kallien G, Lange K, Stange EF, Scheibner J. The pravastatininduced decrease of biliary cholesterol secretion is not directly related to an inhibition of cholesterol synthesis in humans. Hepatology 1999;30:14-20.

15 Miyake JH, Wang SL, Davis RA. Bile acid induction of cytokine expression by macrophages correlates with repression of hepatic cholesterol 7alpha-hydroxylase. J Biol Chem 2000;275:21805-21808.

16 Bertolotti M, Gabbi C, Anzivino C, Mitro N, Godio C, De Fabiani E, et al. Decreased hepatic expression of PPAR-gamma coactivator-1 in cholesterol cholelithiasis. Eur J Clin Invest 2006;36:170-175.

17 Caroli-Bosc FX, Le Gall P, Pugliese P, Delabre B, Caroli-Bosc C, Demarquay JF, et al. Role of fibrates and HMG-CoA reductase inhibitors in gallstone formation: epidemiological study in an unselected population. Dig Dis Sci 2001;46:540-544.

18 Jasińska M, Owczarek J, Orszulak-Michalak D. Statins: a new insight into their mechanisms of action and consequent pleiotropic effects. Pharmacol Rep 2007;59:483-499.

19 Lee J, Shirk A, Oram JF, Lee SP, Kuver R. Polarized cholesterol and phospholipid efflux in cultured gall-bladder epithelial cells: evidence for an ABCA1-mediated pathway. Biochem J 2002;364:475-484.

20 Lee J, Tauscher A, Seo DW, Oram JF, Kuver R. Cultured gallbladder epithelial cells synthesize apolipoproteins A-I and E. Am J Physiol Gastrointest Liver Physiol 2003;285: G630-641.

21 White CM. A review of the pharmacologic and pharmacokinetic aspects of rosuvastatin. J Clin Pharmacol 2002;42: 963-970.

22 Pfefferkorn JA, Song Y, Sun KL, Miller SR, Trivedi BK, Choi C, et al. Design and synthesis of hepatoselective, pyrrole-based HMG-CoA reductase inhibitors. Bioorg Med Chem Lett 2007;17:4538-4544.

23 Hamelin BA, Turgeon J. Hydrophilicity/lipophilicity: relevance for the pharmacology and clinical effects of HMG-CoA reductase inhibitors. Trends Pharmacol Sci 1998;19:26-37.

Received October 20, 2010

Accepted after revision December 30, 2010

Author Affiliations: Division of Gastroenterology, Department of Internal Medicine, Kyung Hee University College of Medicine (Dong SH), and Division of Gastroenterology, Department of Internal Medicine, Hallym University College of Medicine (Lee J, Koh DH, Choi MH, Jang HJ and Kae SH), Seoul 150-030, Republic of Korea

Jin Lee, MD, Division of Gastroenterology, Department of Internal Medicine, Hallym University College of Medicine, Hangang Sacred Heart Hospital, 94-200 Youngdungpo-dong, Youngdungpo-gu, Seoul 150-030, Republic of Korea (Tel: +82-2-2639-5405; Fax: +82-2-2637-9097; Email: jinlee@medimail.co.kr)

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