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• W2012070467 abstract "Inhibition of cholesterol synthesis by 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoAR) inhibitors has been associated with an increase in intestinal cholesterol absorption. This study examined how HMG-CoAR inhibition by atorvastatin modulates expression of key genes involved in intestinal cholesterol metabolism. A crossover study was conducted in which 22 hyperlipidemic men received atorvastatin, 40 mg/day, or placebo, each for 12 weeks. Gene expression was assessed by real-time PCR using duodenal biopsy samples obtained at the end of each phase of treatment. Treatment with atorvastatin was associated with a 76% reduction in lathosterol and significant increases in sitosterol (70%). Atorvastatin significantly increased intestinal mRNA levels of HMG-CoAR (59%), LDL receptor (LDLR) (52%), PCSK9 (187%), SREBP-2 (44%), and HNF-4α (13%). Furthermore, atorvastatin significantly increased intestinal mRNA levels of NPC1L1 by 19% and decreased mRNA levels of both ABCG5 and ABCG8 by 14%. Positive correlations were observed between changes in SREBP-2 and HNF-4α expression and concurrent changes in the intestinal mRNA levels of HMG-CoAR, LDLR, and NPC1L1. These results indicate that HMG-CoAR inhibition with atorvastatin stimulates the intestinal expression of NPC1L1, LDLR, and PCSK9; increases cholesterol absorption; and reduces expression of ABCG5/8; these effects are most likely mediated by upregulation of the transcription factors SREBP-2 and HNF-4α. Inhibition of cholesterol synthesis by 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoAR) inhibitors has been associated with an increase in intestinal cholesterol absorption. This study examined how HMG-CoAR inhibition by atorvastatin modulates expression of key genes involved in intestinal cholesterol metabolism. A crossover study was conducted in which 22 hyperlipidemic men received atorvastatin, 40 mg/day, or placebo, each for 12 weeks. Gene expression was assessed by real-time PCR using duodenal biopsy samples obtained at the end of each phase of treatment. Treatment with atorvastatin was associated with a 76% reduction in lathosterol and significant increases in sitosterol (70%). Atorvastatin significantly increased intestinal mRNA levels of HMG-CoAR (59%), LDL receptor (LDLR) (52%), PCSK9 (187%), SREBP-2 (44%), and HNF-4α (13%). Furthermore, atorvastatin significantly increased intestinal mRNA levels of NPC1L1 by 19% and decreased mRNA levels of both ABCG5 and ABCG8 by 14%. Positive correlations were observed between changes in SREBP-2 and HNF-4α expression and concurrent changes in the intestinal mRNA levels of HMG-CoAR, LDLR, and NPC1L1. These results indicate that HMG-CoAR inhibition with atorvastatin stimulates the intestinal expression of NPC1L1, LDLR, and PCSK9; increases cholesterol absorption; and reduces expression of ABCG5/8; these effects are most likely mediated by upregulation of the transcription factors SREBP-2 and HNF-4α. The relationship between elevated plasma levels of LDL-cholesterol (LDL-C) and the risk of atherosclerosis has been very well established (1Coronary heart disease in seven countries.Circulation. 1970; 41: I1-I211PubMed Google Scholar, 2Castelli W.P. Epidemiology of coronary heart disease: the Framingham study.Am. J. Med. 1984; 76: 4-12Abstract Full Text PDF PubMed Scopus (856) Google Scholar, 3Stamler J. Wentworth D. Neaton J.D. Is relationship between serum cholesterol and risk of premature death from coronary heart disease continuous and graded? Findings in 356,222 primary screenees of the Multiple Risk Factor Intervention Trial (MRFIT).JAMA. 1986; 256: 2823-2828Crossref PubMed Scopus (1772) Google Scholar). Clinical trials have shown that reduction of LDL-C constitutes a primary strategy for the prevention and regression of coronary heart disease (4Genest J. McPherson R. Frohlich J. Anderson T. Campbell N. Carpentier A. Couture P. Dufour R. Fodor G. Francis G.A. 2009 Canadian Cardiovascular Society/Canadian guidelines for the diagnosis and treatment of dyslipidemia and prevention of cardiovascular disease in the adult—2009 recommendations.Can. J. Cardiol. 2009; 25: 567-579Abstract Full Text PDF PubMed Scopus (622) Google Scholar). Plasma cholesterol levels are regulated by feedback mechanisms including exogenous cholesterol absorption through the gastrointestinal tract and endogenous cholesterol synthesis by various tissues. Several studies have shown that the amount of dietary cholesterol absorbed also influences endogenous cholesterol synthesis (5Tilvis R.S. Miettinen T.A. Serum plant sterols and their relation to cholesterol absorption.Am. J. Clin. Nutr. 1986; 43: 92-97Crossref PubMed Scopus (273) Google Scholar, 6Matthan N.R. Raeini-Sarjaz M. Lichtenstein A.H. Ausman L.M. Jones P.J. Deuterium uptake and plasma cholesterol precursor levels correspond as methods for measurement of endogenous cholesterol synthesis in hypercholesterolemic women.Lipids. 2000; 35: 1037-1044Crossref PubMed Scopus (46) Google Scholar, 7Rajaratnam R.A. Gylling H. Miettinen T.A. Independent association of serum squalene and noncholesterol sterols with coronary artery disease in postmenopausal women.J. Am. Coll. Cardiol. 2000; 35: 1185-1191Crossref PubMed Scopus (143) Google Scholar). The newly identified Niemann-Pick C1-like 1 (NPC1L1) protein expressed at the apical membrane of enterocytes has been shown to play a crucial role in the absorption of cholesterol and plant sterol (8Altmann S.W. Davis Jr., H.R. Zhu L.J. Yao X. Hoos L.M. Tetzloff G. Iyer S.P. Maguire M. Golovko A. Zeng M. Niemann-Pick C1 Like 1 protein is critical for intestinal cholesterol absorption.Science. 2004; 303: 1201-1204Crossref PubMed Scopus (1410) Google Scholar). Several physiological determinants and pharmacological agents modulate cholesterol homeostasis, including genetic factors, body weight, ezetimibe therapy, and 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoAR) inhibitors (statins) therapy, the rate-limiting step in the cholesterol biosynthesis pathway (9Dietschy J.M. Turley S.D. Spady D.K. Role of liver in the maintenance of cholesterol and low density lipoprotein homeostasis in different animal species, including humans.J. Lipid Res. 1993; 34: 1637-1659Abstract Full Text PDF PubMed Google Scholar). For instance, obese subjects show an increase in cholesterol synthesis with an associated decrease in cholesterol absorption (10Stahlberg D. Rudling M. Angelin B. Bjorkhem I. Forsell P. Nilsell K. Einarsson K. Hepatic cholesterol metabolism in human obesity.Hepatology. 1997; 25: 1447-1450Crossref PubMed Scopus (76) Google Scholar, 11Miettinen T.A. Gylling H. Cholesterol absorption efficiency and sterol metabolism in obesity.Atherosclerosis. 2000; 153: 241-248Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Ezetimibe therapy has been shown to reduce intestinal cholesterol absorption while reciprocally elevating synthesis (12Sudhop T. Lutjohann D. Kodal A. Igel M. Tribble D.L. Shah S. Perevozskaya I. von Bergmann K. Inhibition of intestinal cholesterol absorption by ezetimibe in humans.Circulation. 2002; 106: 1943-1948Crossref PubMed Scopus (451) Google Scholar). These findings suggest the presence of a reciprocal relationship between cholesterol absorption and synthesis, as a change in one vector results in a compensatory and opposing change in the other. Although recent data suggest that statin therapy is associated with a rise in intestinal cholesterol absorption (13Miettinen T.A. Strandberg T.E. Gylling H. Noncholesterol sterols and cholesterol lowering by long-term simvastatin treatment in coronary patients: relation to basal serum cholestanol.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1340-1346Crossref PubMed Scopus (196) Google Scholar), the impact of HMG-CoAR inhibitors on cholesterol absorption and the molecular mechanisms underlying this effect has not been fully characterized. Therefore, the primary objective of the present study was to gain further insight into this key physiological process by examining the impact of a 12-week regimen of atorvastatin therapy, 40 mg/day, on intestinal expression of the sterol transporter NPC1L1 in subjects with mixed hyperlipidemia. Furthermore, we examined the impact of atorvastatin therapy on intestinal expression of the key gene products involved in cholesterol metabolism, such as ATP-binding cassette transporter 5 (ABCG5) and ABCG8, HMG-CoAR, LDL receptor, sterol regulatory element binding transcription factor 2 (SREBP-2), hepatocyte nuclear factor 4 α (HNF-4α), proprotein convertase subtilisin kexin-9 (PCSK9), and microsomal triglyceride transfer protein (MTTP). Gene expression studies were undertaken using a human duodenal biopsy model, which we have recently developed. Twenty-three men with plasma LDL-C levels above the 50th percentile for their age were recruited from the Quebec City area to participate in the study (14Connelly P.W. MacLean D.R. Horlick L. O'Connor B. Petrasovits A. Little J.A. Plasma lipids and lipoproteins and the prevalence of risk for coronary heart disease in Canadian adults. Canadian Heart Health Surveys Research Group.CMAJ. 1992; 146: 1977-1987PubMed Google Scholar). One subject had to be withdrawn from analyses because of poor RNA quality. Subjects were excluded if they had persistent elevation of serum transaminases; monogenic hyperlipidemia such as familial hypercholesterolemia; plasma triglyceride (TG) levels >4.5 mmol/l; a recent history of alcohol or drug abuse; diabetes mellitus; or a history of cancer. Furthermore, all participants were unrelated at the first and second degree. All eligible subjects had to be withdrawn from lipid-lowering medications for at least 6 weeks before the beginning of the study. The study consisted of a 1 week screening period and a 4 week placebo run-in period, followed by two consecutive 12 week double-blind treatment periods with atorvastatin, 40 mg/day, or placebo in random order. Fasting blood samples and duodenal biopsies were performed following each phase of treatment. Participants were instructed to take one capsule at the time of their evening meal. Compliance was assessed by pill counting. Participants were asked not to change their dietary habits or use of alcohol and level of physical exercise during the study. The research protocol was approved by the Laval University Medical Center ethical review committee, and written informed consent was obtained from each subject. Twelve hour fasting venous blood samples were obtained from an antecubital vein and collected in Vacutainer tubes containing EDTA (0.1%, final concentration) at the end of each phase of treatment. Plasma was separated from blood cells by centrifugation at 3,000 rpm for 10 min at 4°C. Plasma cholesterol and TG concentrations were determined with an Analyzer RA-1000 (Technicon Instruments Corporation, Tarrytown, NY), as previously described (15Moorjani S. Dupont A. Labrie F. Lupien P.J. Brun D. Gagné C. Giguère M. Bélanger A. Increase in plasma high-density lipoprotein concentration following complete androgen blockage in men with prostatic carcinoma.Metabolism. 1987; 36: 244-250Abstract Full Text PDF PubMed Scopus (191) Google Scholar). The LDL-C level was also calculated according to the equation described by Friedewald et al. (16Friedewald W.T. Levy R.I. Fredrickson D.S. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge.Clin. Chem. 1972; 18: 499-502Crossref PubMed Scopus (63) Google Scholar): [LDL-C] = [total cholesterol] − [HDL-C] − [TG]/2.2, and HDL-cholesterol was measured as previously described (17Albers J.J. Warnick G.R. Wiebe D. King P. Steiner P. Smith L. Breckenridge C. Chow A. Kuba K. Weidman S. Multi-laboratory comparison of three heparin-Mn2+ precipitation procedures for estimating cholesterol in high-density lipoprotein.Clin. Chem. 1978; 24: 853-856Crossref PubMed Scopus (150) Google Scholar). Plasma apolipoprotein B-48 (apoB-48) was assessed by ELISA (Shibayagi Co., Japan) (18Kinoshita M. Kojima M. Matsushima T. Teramoto T. Determination of apolipoprotein B-48 in serum by a sandwich ELISA.Clin. Chim. Acta. 2005; 351: 115-120Crossref PubMed Scopus (87) Google Scholar). C-reactive protein (CRP) concentrations were measured with a highly sensitive commercial immunoassay (Dade Behring, Mississauga, ON, Canada) as described previously (19Pirro M. Bergeron J. Dagenais G.R. Bernard P.M. Cantin B. Despres J.P. Lamarche B. Age and duration of follow-up as modulators of the risk for ischemic heart disease associated with high plasma C-reactive protein levels in men.Arch. Intern. Med. 2001; 161: 2474-2480Crossref PubMed Scopus (120) Google Scholar). Plasma concentrations of lathosterol, a precursor in the biosynthesis of cholesterol, and of the plant sterols campesterol and sitosterol, used as plasma surrogates of intestinal cholesterol absorption, were quantified at Laval University, using a gas chromatography method similar to that described previously (20Matthan N.R. Giovanni A. Schaefer E.J. Brown B.G. Lichtenstein A.H. Impact of simvastatin, niacin, and/or antioxidants on cholesterol metabolism in CAD patients with low HDL.J. Lipid Res. 2003; 44: 800-806Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Coefficients of variation ranged between 3.9% and 9.9%. Because non-cholesterol sterols are transported in plasma by lipoproteins, their concentrations have been expressed relative to the concentration of total cholesterol to correct for differing numbers of lipoprotein acceptor particles. This method for quantifying cholesterol absorption has been validated relative to that of the continuous isotope feeding method (21Crouse J.R. Grundy S.M. Evaluation of a continuous isotope feeding method for measurement of cholesterol absorption in man.J. Lipid Res. 1978; 19: 967-971Abstract Full Text PDF PubMed Google Scholar), both in metabolic (22Gylling H. Miettinen T.A. Baseline intestinal absorption and synthesis of cholesterol regulate its response to hypolipidaemic treatments in coronary patients.Atherosclerosis. 2002; 160: 477-481Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) and population settings (23Miettinen T.A. Tilvis R.S. Kesaniemi Y.A. Serum plant sterols and cholesterol precursors reflect cholesterol absorption and synthesis in volunteers of a randomly selected male population.Am. J. Epidemiol. 1990; 131: 20-31Crossref PubMed Scopus (557) Google Scholar). Biopsy samples were obtained from the second portion of the duodenum during gastroduodenoscopy. Six biopsy samples were collected using multiple sample single-use biopsy forceps and immediately flash frozen in liquid nitrogen and stored at −80°C before RNA extraction. Intestinal biopsy tissue samples were homogenized in RLT buffer (Qiagen) using a Tissue-Tearor (BioSpec Products, Inc., Bartlesville, OK) and a 4.5 mm stainless steel probe. The RNA content from homogenized tissue samples were then extracted using an RNeasy fibrous tissue mini-kit (Qiagen). Tissue samples were also treated with an RNase-free DNase set to eliminate any contaminant DNA. Total RNA was then eluted into 100 µl RNase-free H2O and stored at −80°C. RNA quality was assessed with a 2100 Bioanalyzer (Agilent Technologies, Inc.) as previously described (24Luu-The V. Paquet N. Calvo E. Cumps J. Improved real-time RT-PCR method for high-throughput measurements using second derivative calculation and double correction.Biotechniques. 2005; 38: 287-293Crossref PubMed Scopus (231) Google Scholar). Data calculation and normalization were performed using the second derivative and double correction method as described in the study by Warrington et al. (25Warrington J.A. Nair A. Mahadevappa M. Tsyganskaya M. Comparison of human adult and fetal expression and identification of 535 housekeeping/maintenance genes.Physiol. Genomics. 2000; 2: 143-147Crossref PubMed Scopus (447) Google Scholar) and using the reference genes hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1), ATP synthase O subunit (Atp5o), glucose-6-phosphate dehydrogenase (G6PD), and 18S rRNA. The Hprt1, Atp5o, and G6PD genes have been shown to have stable expression levels from embryonic life through adulthood in various tissues. mRNA expression levels are expressed as the number of copies/µg total RNA, using a standard curve of crossing points versus the logarithm of the quantity. The standard curve was established by using known amounts of purified PCR products (10, 102, 103, 104, 105, and 106 copies) and LightCycler 480 version 1.5 software provided by the manufacturer (Roche Inc). Duodenal tissue samples were homogenized, and total protein from each sample was subjected to SDS-PAGE and analyzed by Western blotting (8Altmann S.W. Davis Jr., H.R. Zhu L.J. Yao X. Hoos L.M. Tetzloff G. Iyer S.P. Maguire M. Golovko A. Zeng M. Niemann-Pick C1 Like 1 protein is critical for intestinal cholesterol absorption.Science. 2004; 303: 1201-1204Crossref PubMed Scopus (1410) Google Scholar) with code 1801 polyclonal anti-NPC1L1 antibodies (26Iyer S.P. Yao X. Crona J.H. Hoos L.M. Tetzloff G. Davis Jr., H.R. Graziano M.P. Altmann S.W. Characterization of the putative native and recombinant rat sterol transporter Niemann-Pick C1 Like 1 (NPC1L1) protein.Biochim. Biophys. Acta. 2005; 1722: 282-292Crossref PubMed Scopus (79) Google Scholar). NPC1L1 signal was normalized with the enterocyte-specific marker, villin (8Altmann S.W. Davis Jr., H.R. Zhu L.J. Yao X. Hoos L.M. Tetzloff G. Iyer S.P. Maguire M. Golovko A. Zeng M. Niemann-Pick C1 Like 1 protein is critical for intestinal cholesterol absorption.Science. 2004; 303: 1201-1204Crossref PubMed Scopus (1410) Google Scholar). Plasma PCSK9 was measured by ELISA using a polyclonal antibody against human PCSK9 (27Dubuc G. Tremblay M. Pare G. Jacques H. Hamelin J. Benjannet S. Boulet L. Genest J. Bernier L. Seidah N.G. A new method for measurement of total plasma PCSK9: clinical applications.J. Lipid Res. 2010; 51: 140-149Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Nonparametric Wilcoxon matched pair analyses were used to compare the effects of atorvastatin on the lipid/lipoprotein profile and on mRNA expression. Spearman correlation coefficients were determined to assess the significance of associations. Differences were considered significant at a P value of ≤0.05. All analyses were performed using JMP statistical software (version 8.0.1; SAS Institute, Cary, NC). Participants’ mean ± SD age, body mass index, and waist circumference were 38.1 ± 9.8 years, 29.0 ± 4.0 kg/m2, and 100.1 ± 12.1 cm, respectively. Subjects maintained their weight throughout the study. One subject had to be withdrawn from the analyses because of poor RNA quality. Table 1 shows the lipid/lipoprotein profiles of the 22 subjects following a 12-week treatment with atorvastatin and placebo. Atorvastatin, 40 mg/day, significantly reduced levels of plasma cholesterol (−36.8%; P < 0.0001), LDL-C (−50.0%; P < 0.0001), TG (−28.7%; P = 0.0004), and apoB-48 (−24.2%; P = 0.04) but had no significant effect on plasma HDL-C concentrations and CRP levels. The impact of atorvastatin on plasma surrogates of cholesterol absorption (campesterol and sitosterol) and on synthesis (lathosterol) was also assessed. Compared with placebo, atorvastatin significantly increased plasma campesterol (+64.7%; P < 0.0001) and sitosterol (+69.7%; P < 0.0001) and was associated with a significant reduction in plasma lathosterol (−75.7%; P < 0.0001). The lathosterol/campesterol and lathosterol/sitosterol ratios, representing indexes of cholesterol homeostasis, were significantly reduced following therapy with atorvastatin. Atorvastatin significantly increased plasma levels of PCSK9 (+37.6%; P < 0.0001), a binding protein enhancing the degradation of the LDL receptor in endosomes/lysosomes (28Seidah N.G. PCSK9 as a therapeutic target of dyslipidemia.Expert Opin. Ther. Targets. 2009; 13: 19-28Crossref PubMed Scopus (117) Google Scholar). Interindividual variability in responses of plasma LDL-C, TG, apoB-48, plasma PCSK9, lathosterol, and sitosterol is illustrated in Fig. 1. Twenty-two of 22 participants showed changes in LDL-C, lathosterol, and sitosterol levels in the same direction, while atorvastatin-induced changes in plasma TG and apoB-48 were negative in 18 and 15 subjects, respectively.TABLE 1Lipid/lipoprotein profiles for study subjectsMean ± SD expression level after treatment withCharacteristicPlaceboAtorvastatin% of changePAge (years)38.1 ± 9.838.1 ± 9.8Body mass index (kg/m2)29.0 ± 4.029.0 ± 4.0Waist circumference (cm)100.1 ± 12.1100.1 ± 12.1Plasma-cholesterol (mmol/l)5.72 ± 0.953.61 ± 0.48−36.8<0.0001Triglycerides (mmol/l)1.57 ± 0.811.12 ± 0.54−28.70.0004LDL-cholesterol (mmol/l)3.86 ± 0.841.93 ± 0.45−50.0<0.0001HDL-cholesterol (mmol/l)1.14 ± 0.241.17 ± 0.24+2.60.15Plasma apolipoprotein B-48 (ng/ml)9571 ± 54507252 ± 5876−24.20.04C-reactive protein (mg/l)2.50 ± 2.202.24 ± 2.18−10.40.31PCSK9 (ng/ml)62.8 ± 13.486.4 ± 22.7+37.6<0.0001Plasma lathosterol231.4 ± 110.556.3 ± 22.3−75.7<0.0001Plasma campesterol157.7 ± 73.6259.8 ± 83.9+64.7<0.0001Plasma sitosterol158.6 ± 72.0269.1 ± 80.6+69.7<0.0001Lathosterol/campesterol ratio1.94 ± 1.370.25 ± 0.15−87.1<0.0001Lathosterol/sitosterol ratio1.95 ± 1.440.23 ± 0.11−88.4<0.0001Table shows lipid/lipoprotein profiles and surrogate markers of cholesterol synthesis and absorption from 22 subjects following 12-week treatment with atorvastatin, 40 mg/day. P values are the difference between baseline and treatment values. PCSK9, proprotein convertase subtilisin kexin-9. Open table in a new tab Table shows lipid/lipoprotein profiles and surrogate markers of cholesterol synthesis and absorption from 22 subjects following 12-week treatment with atorvastatin, 40 mg/day. P values are the difference between baseline and treatment values. PCSK9, proprotein convertase subtilisin kexin-9. As shown in Table 2, studies of gene product expression revealed that atorvastatin significantly increased intestinal mRNA levels of HMG-CoAR (+59.4%; P < 0.0001), LDL receptor (+52.2%; P = 0.0007), acetyl-CoA acetyltransferase-2 (ACAT-2) (+64.5%; P < 0.0001), SREBP-2 (+44.4%; P < 0.0001), and HNF-4α (+13.4%; P = 0.02). Intestinal mRNA expression levels of PCSK9 (+186.6%; P < 0.0001) and NPC1L1 (+18.7%, P=0.03) were also significantly increased by atorvastatin. On the other hand, atorvastatin decreased mRNA expression levels of ABCG5 and ABCG8 by −14.0% (P = 0.04) and −13.6% (P = 0.06), respectively. Finally, treatment with atorvastatin had no significant impact on mRNA levels of apoB-48, fatty acid binding protein-2 (FABP-2), fatty acid transporter protein-4 (FATP-4), MTTP, and SREBP-1c. As shown in Fig. 2, the atorvastatin-induced changes in mRNA levels of HMG-CoAR, LDL receptor, and SREBP-2 were positive in at least 19 of 22 participants, while 14 participants exhibited changes in NPC1L1 and ABCG5/8 expression in the same direction.TABLE 2Intestinal gene mRNA expression levelsMean no. of copies ± SD/100,000 copies of Atp5o control gene followingGenePlaceboAtorvastatin% of changePABCG515,393 ± 4,95913,241 ± 7,004−14.00.04ABCG82,959 ± 1,1602,558 ± 1208−13.60.06ACAT-21,756 ± 4542,888 ± 889+64.5<0.0001ApoB gene66,611 ± 30,68675,654 ± 32,739+13.60.1FABP-221,250 ± 6,64922,043 ± 7,126+3.7NSFATP-412,475 ± 2,02112,943 ± 2,125+3.8NSHMG-CoAR2,866 ± 6394,568 ± 952+59.4<0.0001HNF-4α9,558 ± 2,26910,841 ± 2,402+13.40.02LDL receptor3,915 ± 2,2935,959 ± 1875+52.20.0007MTTP134,810 ± 34,405136,137 ± 34,707+1.0NSNPC1L19,376 ± 2,78111,127 ± 2,919+18.70.03PCSK9238 ± 174682 ± 298+186.6<0.0001SREBP-1c3,951 ± 1,0003,806 ± 987−3.7NSSREBP-22,215 ± 5333,199 ± 745+44.4<0.0001NS, not significant; ABCG5/8, ATP binding cassette; ACAT-2, acetyl-CoA acetyltransferase 2; FABP-2, fatty acid binding protein 2; FATP-4, fatty acid transporter protein 4; HMG-CoAR, 3-hydroxyl-3-methylglutaryl-CoA reductase; HNF-4α, hepatocyte nuclear factor-4α; MTTP, microsomal triglyceride transfer protein; NPC1L1, Niemann-Pick C1-like 1; PCSK9, proprotein convertase subtilisin kexin-9; SREBP-1c and -2, sterol regulatory element binding transcription factors 1c and 2. Open table in a new tab NS, not significant; ABCG5/8, ATP binding cassette; ACAT-2, acetyl-CoA acetyltransferase 2; FABP-2, fatty acid binding protein 2; FATP-4, fatty acid transporter protein 4; HMG-CoAR, 3-hydroxyl-3-methylglutaryl-CoA reductase; HNF-4α, hepatocyte nuclear factor-4α; MTTP, microsomal triglyceride transfer protein; NPC1L1, Niemann-Pick C1-like 1; PCSK9, proprotein convertase subtilisin kexin-9; SREBP-1c and -2, sterol regulatory element binding transcription factors 1c and 2. As shown in Fig. 3, changes in intestinal mRNA levels of ABCG5 were significantly correlated with changes in mRNA levels of ABCG8, while changes in HNF-4α expression were highly and positively correlated with changes in mRNA levels of SREBP-2. Fig. 4 shows positive correlations between changes in SREBP-2 mRNA levels and concurrent changes in mRNA levels of HMG-CoAR (r = 0.45; P = 0.04), LDL receptor (r = 0.59; P = 0.004), and NPC1L1 (r = 0.65; P = 0.0007). In addition, positive correlations were also observed between changes in HNF-4α mRNA levels and concurrent changes in mRNA levels of HMG-CoAR (r = 0.45; P = 0.03), LDL receptor (r = 0.57; P=0.005), and NPC1L1 (r = 0.66; P=0.0008).Fig. 4Correlation between changes in HMG-CoAR, LDL receptor, and intestinal NPC1L1 mRNA expression and changes in intestinal SREBP-2 (A) and HNF-4α (B) mRNA expression following treatment with atorvastatin, 40 mg/day, versus placebo are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Following treatment with atorvastatin, subjects’ mean percentage of changes in NPC1L1 protein expression in intestinal biopsy samples was increased by +33.5%, but this difference did not reach statistical significance due to a large variability in protein measurements. In addition, no significant correlation was observed between changes in NPC1L1 protein levels and changes in mRNA levels of NPC1L1 (data not shown). In the present study, the 12-week treatment with atorvastatin, 40 mg/day, resulted in a significant reduction in levels of plasma cholesterol (−37%), TG (−29%), LDL-C (−50%), and apolB-48 (−24%). Furthermore, atorvastatin significantly decreased plasma lathosterol (−76%), a marker of cholesterol synthesis, and significantly increased plasma campesterol (+65%) and sitosterol (+70%), two surrogates of cholesterol absorption. Finally, treatment with atorvastatin upregulated intestinal mRNA levels of NPC1L1 (+19%), HMG-CoAR (+59%), LDL receptor (52%), ACAT-2 (+65%), SREBP-2 (+44%), HNF-4α (+13%), and PCSK9 (+187%) and reduced ABCG5/8 expression by 14%. The homeostasis of circulating cholesterol levels is modulated primarily by cholesterol absorption and synthesis (29Dietschy J.M. Siperstein M.D. Effect of cholesterol feeding and fasting on sterol synthesis in seventeen tissues of the rat.J. Lipid Res. 1967; 8: 97-104Abstract Full Text PDF PubMed Google Scholar, 30Spady D.K. Dietschy J.M. Sterol synthesis in vivo in 18 tissues of the squirrel monkey, guinea pig, rabbit, hamster, and rat.J. Lipid Res. 1983; 24: 303-315Abstract Full Text PDF PubMed Google Scholar). Several factors have been shown to influence cholesterol homeostasis including genetic factors, circadian rhythm, body weight, and various therapeutic agents such as ezetimibe, statins, and plant sterols (9Dietschy J.M. Turley S.D. Spady D.K. Role of liver in the maintenance of cholesterol and low density lipoprotein homeostasis in different animal species, including humans.J. Lipid Res. 1993; 34: 1637-1659Abstract Full Text PDF PubMed Google Scholar). Recent data have suggested that the downregulation of cholesterol synthesis by statin therapy is compensated by a rise in intestinal cholesterol absorption (11Miettinen T.A. Gylling H. Cholesterol absorption efficiency and sterol metabolism in obesity.Atherosclerosis. 2000; 153: 241-248Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Our study is consistent with this concept (31Ntanios F.Y. Jones P.J. Frohlich J.J. Effect of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor on sterol absorption in hypercholesterolemic subjects.Metabolism. 1999; 48: 68-73Abstract Full Text PDF PubMed Scopus (11) Google Scholar, 32Miettinen T.A. 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: 976-982Crossref PubMed Scopus (119) Google Scholar), having shown a reduction in plasma lathosterol levels (synthesis) compensated by an increase in both campesterol and sitosterol (absorption) following treatment with atorvastatin. In the present study, treatment with atorvastatin significantly increased intestinal mRNA levels of NPC1L1, which was paralleled by a nonsignificant increase in NPC1L1 protein expression. In agreement with our findings, a study with miniature pigs showed that NPC1L1 expression was increased incrementally in both the jejunum and the liver by combination therapy with ezetimibe and simvastatin (33Telford D.E. Sutherland B.G. Edwards J.Y. Andrews J.D. Barrett P.H. Huff M.W. The molecular mechanisms underlying the reduction of LDL apoB-100 by ezetimibe plus simvastatin.J. Lipid Res. 2007; 48: 699-708Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Activation of the nuclear transcription factor SREBP-2 is known to be negatively regulated by sterols and was recently reported to activate NPC1L1 transcription (34Alrefai W.A. Annaba F. Sarwar Z. Dwive" @default.
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• W2012070467 title "Atorvastatin increases intestinal expression of NPC1L1 in hyperlipidemic men" @default.
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