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Human Nutrition Research Center of Nantes, France, * Endocrinology and Nutrition Unit, National Veterinary School of Nantes, France, and INSERM U539, University Hospital, Nantes, France
3 To whom correspondence should be addressed. E-mail: pnguyen{at}vet-nantes.fr.
KEY WORDS: • atorvastatin • cholesterol plasma levels • sterol absorption
Statins are widely used to reduce low-density lipoprotein cholesterol levels and associated cardiovascular events (1). However, intestinal cholesterol absorption is increased in humans treated with statins (2). Inhibition of intestinal sterols absorption therefore represents a new therapy to reduce cholesterol plasma levels (3), but understanding the mechanism involved in sterol absorption needs investigation.
Transgenic and knock-out mice have been widely used to study the mechanisms and receptors involved in cholesterol absorption. In this animal model, Niemann-Pick C1 Like 1 (NPC1L1)4 protein was discovered as the intestinal phytosterol and cholesterol transporter (4), and has been identified as the target of the cholesterol absorption inhibitor ezetimibe (5). Adenosine triphosphate-binding cassette (ABC) transporters, such as ABCA1, ABCG5, and ABCG8, are also gaining potent interest because activation of the nuclear receptor liver X receptor upregulates expression of these transporters and finally inhibits intestinal absorption of cholesterol (6,7). Scavenger receptor class B type I (SR-BI) has been localized on both apical and basolateral membranes of intestinal cells (8), but the role of this receptor in cholesterol absorption is unclear.
Very recently, cholesterol transport in mice has been classified into 2 independently modulated, apolipoprotein (apo) B-dependent and apoB-independent, pathways (9). The ApoB-dependent pathway is represented by the chylomicron assembly, mediated by microsomal transfert protein (MTP). MTP activity is essential for the packaging of apolipoprotein and lipids to form chylomicron (10,11). The apoB-independent pathway involves intestinal apoA-I-containing high density lipoproteins (HDL) and cholesterol efflux mediated by the ABCA1 (12). This pathway would represent 25–30% of the total cholesterol absorption in mice (10).
Overall, mechanisms of cholesterol absorption have been mainly studied in rodent models. There is little information, however, about this process in larger animal models such as dog. Because dogs have been used to demonstrate the potent hypocholesterolemic effect of statins and the cholesterol absorption inhibitor ezetimibe (13), the dog is a relevant model for investigations of lipid metabolism in obesity and insulin resistance (14). Therefore, there is a great interest in determining the mechanisms involved in intestinal cholesterol absorption in the dog. In the present study, the effects of atorvastatin on cholesterol absorption in dogs were assessed with a dual stable isotope method. To get further insights into the mechanisms of intestinal cholesterol absorption, we also measured MTP activity and SR-BI expression before and after atorvastatin treatment.
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MATERIALS AND METHODS |
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Percent cholesterol absorption was assessed using the dual stable isotope method, as previously described (15), which was based on the simultaneous administration of [2,2,3,4,4,6-2H6] cholesterol ([2H6] cholesterol) given orally and [25,26,26,26,27,27,27-2H7] cholesterol ([2H7]cholesterol) given intravenously. Plasma cholesterol isotope ratio is measured at a set point in time, with i.v. dose assumed as a "100% absorption" reference. [2H6] Cholesterol and [2H7] cholesterol were obtained from Eurisotop. [2H6] Cholesterol for oral administration was completely dissolved in sunflower oil (16 mg/ml) by rotating overnight at room temperature and 16 mg were placed into the daily meal of each dog. [2H7] Cholesterol for i.v. administration was dissolved in ethanol at 10 mg/mL and filtered through a 0.2 µm solvent-resistant filter (Millex-FG, Millipore). The ethanolic cholesterol tracer was added drop wise to 4 volumes of freshly opened Endolipide 20% (Braun Medical) and gently mixed. Total dissolution of the tracer was checked by confocal microscopy. The day of the experiment, fasted dogs received 8 mg of [2H7] cholesterol through an i.v. NaCl 0.9% sterile infusion for 10 min. The syringe was washed twice with NaCl 0.9% solution. Immediately after i.v. administration, dogs consumed a normal meal containing 16 mg of [2H6] cholesterol. Fasting plasma sample for cholesterol enrichment was drawn before and 72 h after the test.
Nonsaponifiable sterols were extracted into hexane after saponification of 0.5 mL of plasma and derivatized with N-methyl-N-trimethylsilyltrifluoroacetamide (60°C, 30 min).
Electron-impact gas chromatography-mass spectrometry was performed on a 5891A gas chromatograph connected with a 5971A quadrupole mass spectrometer (Hewlett-Packard). Because the principal molecular ion of cholesterol at m/z 458 is too intense, ions were monitored over time at m/z 459 (natural cholesterol), 464 ([2H6] cholesterol), and 465 ([2H7] cholesterol). To determine the molar ratio of [2H6] cholesterol:[2H7] cholesterol, a standard curve was determined by plotting the area ratio 464:465 against weighed mole ratios of [2H6] cholesterol:[2H7] cholesterol diluted in free cholesterol. Percent cholesterol absorption was then calculated as the mole ratio of tracers in plasma at t = 72 h divided by the mole ratio of tracers administered.
Total cholesterol, triglycerides, and phospholipids were measured in plasma and duodenum samples using enzymatic methods (BioMérieux). Each duodenum sample was weighed and homogenized with 1x PBS. Lipids in homogenate were extracted using diisopropylether-butanol (6:4 v:v). The lipid extract was evaporated under a stream of nitrogen gas and redissolved in absolute ethanol for lipids measurement, expressed as mg/g of tissue. MTP activity was measured using a kit (Roarbiomedical).
Duodenum pieces were homogenized in 1x PBS containing 0.25% Na-deoxycholate, 1% Triton X-100 and protease inhibitor cocktail (Roche Diagnostics). The supernatant was collected and 50 µg of duodenum protein was resolved on Nu-PAGE 4–12% Bis-Tris gels in MES-SDS buffer (Invitrogen) under reducing conditions. Protein concentration was determined using the bicinchoninic acid protein assay kit (Interchim). Proteins were transferred onto a protran nitrocellulose membrane (Schleicher and Shuell), probed with polyclonal rabbit antiscavenger receptor class B type I (SR-BI; Novus) or polyclonal rabbit anti-apoA-I (Calbiochem-Novabiochem), using Vectastain PK6101 (AbCyse). For each antibody, cross reactivity against canine apoA-I or canine SR-BI was tested, using human plasma HDL or mouse liver, respectively.
Results are expressed as means ± SEM. Statistical analysis using StatView 5.0 (SAS) was performed with paired t-test to determine significant differences between variables for dogs before and after atorvastatin treatment. A 2-sided P < 0.05 was considered significant.
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RESULTS |
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TOPMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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DISCUSSION |
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TOPMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Higher cholesterol absorption was reported in humans treated with atorvastatin at 80 mg/d (2). However, this effect was measured with plant sterols as markers of cholesterol absorption, and the method was limited because plasma plant sterol concentration depends on the diet (16). We therefore used the dual isotope method to assess a higher percentage of cholesterol absorption with atorvastatin treatment in dogs. The mean percentage of cholesterol absorption before atorvastatin treatment (74.9 ± 2.9%) was close to the 73% value previously reported in dogs using the same method (17). The present results, therefore, strengthen the accuracy of the dual isotope method used to measure cholesterol absorption and its modulation by pharmacologic or nutritional factors.
Because the mechanisms involved in the upregulation of cholesterol absorption with statin treatment remain unclear, we investigated the role of both apoB-dependent and apoB-independent pathways in intestinal cells. Although there were no changes in phospholipid or triglyceride concentrations, atorvastatin treatment reduced cholesterol concentrations in the duodenum. This did not affect MTP activity, and a role for the apoB-dependent pathway would therefore be unlikely, because MTP has been recognized as a central player in this pathway in mouse and cellular models (11,12). Upregulation of cholesterol absorption during atorvastatin treatment may therefore occur through the apoB-independent pathway that involves apoA-I, ABCA1, and intestinal HDL. However, a higher apoA-I expression in the intestine during atorvastatin treatment was the only piece of evidence for this theoretical possibility. This result is consistent with the increased HDL secretion that has been observed when free apoA-I is supplied to isolated enterocytes (10).
Although SR-BI would be involved in enterocyte lipid trafficking (18), its expression did not change despite higher cholesterol absorption and decreased cellular cholesterol levels. Because cholesterol absorption did not decrease in SR-BI knock-out mice (19,20), the receptor was described as not essential in intestinal cholesterol absorption, and this was demonstrated again in our study. Reduced cholesterol content activates other receptors that are involved in cholesterol absorption, namely, NPC1L1 and ABCA1 proteins. In mice, NPC1L1 and ABCA1 expression was shown to be downregulated by elevated cellular cholesterol concentrations (4,21). Moreover, ABCA1 is involved in the apoB-independent pathway and contributes to HDL-apoA-I secretion by the intestine (12). Upregulation of NPC1L1 and ABCA1 expression would therefore explain higher cholesterol absorption with atorvastatin, but this requires further study.
We conclude that atorvastatin treatment increases cholesterol absorption in dogs, using a dual stable isotope method, even though we did not use control dogs in this study. Our data suggests that increased absorption occurs through an apoB-independent pathway. Although further investigations are needed to describe the cellular mechanism of cholesterol transfer from intestinal lumen to the secretion of chylomicron, the dog could be a relevant model to study nutritional and pharmacological effects on cholesterol absorption. The combination of lipid lowering drugs (e.g., statins and fibrates) and cholesterol absorption inhibitors represents an exciting strategy for the prevention of coronary artery disease. Because the inhibition of apoB-dependent pathways through MTP antagonists has not been successful (11), an understanding of the apoB-independent pathway will be important in developing dyslipidemia treatment with cholesterol absorption inhibition.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Author disclosure: Expenses for authors (F.B. and P.N.) to travel to the symposium were paid by Royal Canin.
4 Abbreviations used: ABC, adenosine triphosphate-binding cassette; apo, apolipoprotein; MTP, microsomal transfer protein; NPC1L1, Niemann-Pick C1 like 1 protein; SR-BI, scavenger receptor class B type I.
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LITERATURE CITED |
|---|
|
TOPMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
1. Schaefer EJ, Brousseau ME. Perspectives: benefits of reducing low-density lipoprotein cholesterol concentrations to <100 mg/dL. Prev Cardiol. 2000;3:136–9.[Medline]
2. Miettinen TA, Gylling H. Synthesis and absorption markers of cholesterol in serum and lipoproteins during a large dose of statin treatment. Eur J Clin Invest. 2003;33:976–82.[Medline]
3. Sudhop T, Lutjohann D, von Bergmann K. Sterol transporters: targets of natural sterols and new lipid lowering drugs. Pharmacol Ther. 2005;105:333–41.[Medline]
4. Davis HR, Jr., Zhu LJ, Hoos LM, Tetzloff G, Maguire M, Liu J, Yao X, Iyer SP, Lam MH, et al. Niemann-Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole-body cholesterol homeostasis. J Biol Chem. 2004;279:33586–92.
5. Garcia-Calvo M, Lisnock J, Bull HG, Hawes BE, Burnett DA, Braun MP, Crona JH, Davis HR, Jr., Dean DC, et al. The target of ezetimibe is Niemann-Pick C1-Like 1 (NPC1L1). Proc Natl Acad Sci USA. 2005;102:8132–7.
6. Knight BL, Patel DD, Humphreys SM, Wiggins D, Gibbons GF. Inhibition of cholesterol absorption associated with a PPAR alpha-dependent increase in ABC binding cassette transporter A1 in mice. J Lipid Res. 2003;44:2049–58.
7. Kaneko E, Matsuda M, Yamada Y, Tachibana Y, Shimomura I, Makishima M. Induction of intestinal ATP-binding cassette transporters by a phytosterol-derived liver X receptor agonist. J Biol Chem. 2003;278:36091–8.
8. Cai SF, Kirby RJ, Howles PN, Hui DY. Differentiation-dependent expression and localization of the class B type I scavenger receptor in intestine. J Lipid Res. 2001;42:902–9.
9. Iqbal J, Hussain MM. Evidence for multiple complementary pathways for efficient cholesterol absorption in mice. J Lipid Res. 2005;46:1491–501.
10. Iqbal J, Anwar K, Hussain MM. Multiple, independently regulated pathways of cholesterol transport across the intestinal epithelial cells. J Biol Chem. 2003;278:31610–20.
11. Hussain MM, Kancha RK, Zhou Z, Luchoomun J, Zu H, Bakillah A. Chylomicron assembly and catabolism: role of apolipoproteins and receptors. Biochim Biophys Acta. 1996;1300:151–70.[Medline]
12. Hussain MM, Fatma S, Pan X, Iqbal J. Intestinal lipoprotein assembly. Curr Opin Lipidol. 2005;16:281–5.[Medline]
13. Davis HR Jr., Pula KK, Alton KB, Burrier RE, Watkins RW. The synergistic hypocholesterolemic activity of the potent cholesterol absorption inhibitor, ezetimibe, in combination with 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitors in dogs. Metabolism. 2001;50:1234–41.[Medline]
14. Bailhache E, Nguyen P, Krempf M, Siliart B, Magot T, Ouguerram K. Lipoproteins abnormalities in obese insulin-resistant dogs. Metabolism. 2003;52:559–64.[Medline]
15. Bosner MS, Lange LG, Stenson WF, Ostlund RE, Jr. Percent cholesterol absorption in normal women and men quantified with dual stable isotopic tracers and negative ion mass spectrometry. J Lipid Res. 1999;40:302–8.
16. Vanstone CA, Jones PJ. Limitations of plasma plant sterols as indicators of cholesterol absorption. Am J Clin Nutr. 2004;79:340–1.
17. Melchior GW, Harwell JF. Cholesterol absorption and turnover in hypercholesterolemic dogs. J Lipid Res. 1985;26:306–15.[Abstract]
18. Hansen GH, Niels-Christiansen LL, Immerdal L, Danielsen EM. Scavenger receptor class B type I (SR-BI) in pig enterocytes: trafficking from the brush border to lipid droplets during fat absorption. Gut. 2003;52:1424–31.
19. van Bennekum A, Werder M, Thuahnai ST, Han CH, Duong P, Williams DL, Wettstein P, Schulthess G, Phillips MC, et al. Class B scavenger receptor-mediated intestinal absorption of dietary beta-carotene and cholesterol. Biochemistry. 2005;44:4517–25.[Medline]
20. Mardones P, Quinones V, Amigo L, Moreno M, Miquel JF, Schwarz M, Miettinen HE, Trigatti B, Krieger M, et al. Hepatic cholesterol and bile acid metabolism and intestinal cholesterol absorption in scavenger receptor class B type I-deficient mice. J Lipid Res. 2001;42:170–80.
21. Repa JJ, Turley SD, Lobaccaro JA, Medina J, Li L, Lustig K, Shan B, Heyman RA, Dietschy JM, et al. Regulation of absorption and ABC1-mediated efflux of cholesterol by RXR heterodimers. Science. 2000;289:1524–29.
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