Reduction of Cholesterol Absorption by Dietary Plant Sterols and Stanols in Mice Is Independent of the Abcg5/8 Transporter1,2
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The Journal of Nutrition Biochemical, Molecular, and Genetic Mechanisms Reduction of Cholesterol Absorption by Dietary Plant Sterols and Stanols in Mice Is Independent of the Abcg5/8 Transporter1,2 Torsten Plo¨ sch,3,4* Janine K. Kruit,3,4 Vincent W. Bloks,4 Nicolette C. A. Huijkman,4 Rick Havinga,4 Guus S. M. J. E. Duchateau,5 Yuguang Lin,5 and Folkert Kuipers4 4Center for Liver, Digestive and Metabolic Diseases, Laboratory of Pediatrics, University Medical Center Groningen, Groningen, The Netherlands and 5Unilever Food and Health Research Institute, Vlaardingen, The Netherlands Downloaded from https://academic.oup.com/jn/article/136/8/2135/4664773 by guest on 25 September 2021 Abstract Dietary supplementation with plant sterols, stanols, and their esters reduces intestinal cholesterol absorption, thus lowering plasma LDL cholesterol concentration in humans. It was suggested that these beneficial effects are attributable in part to induction of genes involved in intestinal cholesterol transport, e.g., Abcg5 and Abcg8, via the liver X receptor (LXR), but direct proof is lacking. Male C57BL/6J mice were fed a purified diet (control), diets containing cholesterol (0.12 g/100 g) only, or in combination with either plant sterols or stanols (0.5 g/100 g) for 4 wk. Plant sterols and stanols dramatically increased neutral fecal sterol excretion (2.2 and 1.4-fold, respectively, compared with cholesterol-fed mice; P , 0.05). Cholesterol and cholesterol ester concentrations were higher in livers of mice fed cholesterol compared with controls (1135% and 1925%; P , 0.05). Plant sterols and stanols completely prevented cholesterol accumulation as well as induction of LXR target genes in liver. Feeding plant sterols and stanols did not alter intestinal expression of Abcg5, Abcg8, or other LXR target genes nor of Npc1l1. Fractional cholesterol absorption in Abcg52/2 mice was reduced to the same extent by dietary plant sterols (49%) as in wild-type littermates (44%). Plant sterol and stanol-induced reduction of cholesterol absorption in mice is not associated with upregulation of intestinal LXR target genes nor is it influenced by Abcg5-deficiency. Our data indicate that dietary plant sterols and stanols inhibit cholesterol absorption within the intestinal lumen independently of LXR. J. Nutr. 136: 2135–2140, 2006. Introduction taken up by enterocytes via NPC1L1 (7). In the enterocyte, Dietary plant sterols and plant stanols have been recognized as cholesterol is readily esterified by the action of acyl-CoA:cho- efficient modulators of plasma LDL cholesterol concentrations lesterol acyltransferase 2 (ACAT2) and released into lymph in in humans for decades (1,2). In contrast to cholesterol, these association with chylomicrons. In contrast, plant sterols and compounds are only poorly absorbed from the intestine. Based stanols are effectively excreted back to the intestinal lumen by on their structural similarity to cholesterol, it was postulated heterodimers of the ATP-binding cassette transporters, ABCG5 that plant sterols and plant stanols physically interfere with and ABCG8, present at the apical membrane of the enterocyte intestinal cholesterol absorption, e.g., by interference with (8,9). ABCA1 is also highly expressed in the intestine, mainly at cholesterol micellization in the intestinal lumen (3–5). the basolateral domain of the cell membrane, but its role in Recently, proteins involved in cholesterol absorption as well sterol absorption is unclear (10–12). Expression of ABCA1, as in intestinal sterol excretion were identified. Niemann-Pick ABCG5, and ABCG8, but not of NPC1L1, is under control of a C1-Like protein 1 (NPC1L1)6 is considered the target of the nuclear receptor, the liver-X-receptor (LXR), activated by cholesterol absorption inhibitor ezetimibe and was shown to be oxysterols (13,14). Activation of LXR by synthetic ligands in responsible for the majority of cholesterol uptake into entero- mice was shown to increase fecal neutral sterol loss and to cytes (6,7). Recent findings indicate that plant sterols are also reduce fractional cholesterol absorption, as a consequence of increased Abcg5/Abcg8 expression, leading to enhanced choles- 1 Supported in part by Unilever Food and Health Research Institute, Vlaardingen, terol excretion back into the intestinal lumen (14,15). The Netherlands and the Dutch Heart Foundation, grant 2004T048 to T.P. and The discovery that cholesterol absorption can be actively 2001B043 to J.K.K. 2 Supplemental Tables 1 and 2 are available with the online posting of this regulated at the level of the enterocyte opens the possibility that paper at jn.nutrition.org. plant sterols and plant stanols, in addition to their postulated 3 These authors contributed equally. physicochemical effects (3–5), also influence cholesterol absorp- 6 Abbreviations used: Abca1, Abcg5, Abcg8: ATP-binding cassette transporter tion by regulating expression of transport proteins. Specifically, a1, g5, g8; Acat2: acyl-CoA:cholesterol acyltransferase 2; LXR: liver-X-receptor; plant sterols and stanols may act as LXR agonists either directly Npc1l1: Niemann-Pick C1-Like 1 protein. * To whom correspondence should be addressed. E-mail: t.plosch@med. or after their conversion into oxyphytosterols or oxyphytostanols, umcg.nl. respectively. Plant sterols and stanols were shown to act as LXR 0022-3166/06 $8.00 ª 2006 American Society for Nutrition. 2135 Manuscript received 27 February 2006. Initial review completed 24 April 2006. Revision accepted 11 May 2006. activators in in vitro experiments (16,17) and for very specific TABLE 1 Composition of purified diets1–3 sterols, also in vivo in mice (18). Cell lines frequently lack ABCG5/ABCG8 expression and therefore may accumulate Control Cholesterol Plant sterol Plant stanol T0901317 higher plant sterol and stanol levels than enterocytes in vivo. g/kg To test whether plant sterols and stanols present in functional food items are able to induce LXR-activated gene expression Cholesterol - 1.20 1.20 1.20 - in vivo, we fed C57BL/6 mice a purified diet virtually free of Plant sterol esters - - 8.33 - - cholesterol and the same diet enriched with cholesterol (0.12 g/ Plant stanol esters - - - 8.33 - 100 g), cholesterol and plant sterol fatty acid esters (0.12 and T0901317 - - - - 0.150 0.83 g/100g), or cholesterol and plant stanol fatty acid esters 1 All diets contained (g/kg): calcium caseinate, 161.40; vitamin mix, 11.30; mineral (0.12 and 0.83 g/100 g) for 4 wk. The plant sterol and stanol mix, 39.70; Arbocel BC-200, 56.70; fat (soybean oil), 126.10; carbohydrates (corn), fatty acid ester doses were equivalent to 0.5 g/100 g sterol or 599.90; L-cysteine HCl, 2.00; choline bitartrate, 2.80. stanol, respectively. A purified diet with the established LXR 2 The purified diets were provided by Unilever (Vlaardingen, The Netherlands). agonist T0901317 (0.015 g/100 g) was used as a positive control Analysis of sterol composition revealed that the control diet contained 0.03% (wt/wt) sterols, mainly b-sitosterol (53.8% of all sterols). Plant sterol fatty acid for gene expression studies (19). Furthermore, we measured esters were mainly b-sitosterol, campesterol, and stigmasterol (36.5, 20.4 and Downloaded from https://academic.oup.com/jn/article/136/8/2135/4664773 by guest on 25 September 2021 intestinal gene expression profiles and fractional cholesterol 14.9%, respectively). Plant stanol esters were mainly b-sitostanol and campestanol absorption in Abcg5 knock-out mice and their wild-type (50.3 and 24.6%). The plant sterol or stanol ester dose is equivalent to 5 g of sterol littermates fed both the cholesterol diet and the cholesterol or stanol/kg diet. 3 diet enriched with plant sterols. Enterocytes in Abcg5 knockout Vitamin mix (g/kg mix): nicotinic acid, 3; Ca pantothenate, 1.6; pyridoxine-HCl, 0.7; thiamine-HCl, 0.6; riboflavin, 0.6; folic acid, 0.2; biotin, 0.02; cyanocobalamin, 5; mice are not protected against plant sterol accumulation; as a all-rac-a-tocopheryl acetate (50%), 15; all-trans-retinyl palmitate, 0.8; cholecalciferol, consequence, they should be more susceptible to LXR-mediated 1; phylloquinone, 0.1; cornstarch, 971.38. The AIN-93G mineral mix contained (g/kg activation of gene expression by plant sterols. mix): calcium carbonate, 236.91; potassium hydrogen phosphate, 196; sodium The aim of this study was to determine whether the well- chloride, 74; magnesium oxide, 24; potassium citrate, 70.78; potassium sulfate, 46.6; AIN mineral mix, 91.71; cornstarch, 260 (38). characterized inhibitory action of plant sterols and plant stanols on intestinal cholesterol absorption is dependent on the activa- tion of LXR in different mouse models. Life Science) dissolved in Intralipid (20%; Fresenius Kabi) and an oral dose of 1.2 mCi of 14C-cholesterol (Amersham Bioscience) dissolved in medium-chain triglyceride oil. 14C and 3H activity was measured by Materials and Methods liquid scintillation counting. Blood samples obtained by retroorbital puncture 48 h after administration were used for the calculation of Animal experiments. Male, 3-mo-old C57BL/6J mice were purchased cholesterol absorption. from Harlan. Mice were housed in temperature-controlled rooms (21°C) with 12-h light cycling and consumed a purified diet and water ad RNA isolation and PCR procedures. Total RNA was extracted from libitum. The composition of the purified diet (prepared by Unilever) is frozen tissues with TriReagent (Sigma) and quantified photometrically. given in Table 1. All mice were fed the purified control diet for 2 wk (run-in cDNA synthesis was performed using recombinant M-MLV reverse period); then, they were assigned to 1 of 5 treatment groups (n ¼ 6) based transcriptase (10 U/mL), the appropriate buffer, dNTPs (500 mmol/L), on their body weights and were fed the specific diets (control, cholesterol random nonamers (1 mmol/L), RNAse inhibitor (2 U/mL; all from Sigma) diet, plant sterol diet, plant stanol diet, T0901317 diet) for 4 wk.