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Are LXR-regulated genes a major molecular target of plant sterols/stanols?

      We share other authors’ interest in the mechanism by which plant sterols reduce intestinal cholesterol absorption and by the molecular actions of phytosterols [
      • Calpe-Berdiel L.
      • Escola-Gil J.C.
      • Ribas V.
      • et al.
      Changes in intestinal and liver global gene expression in response to a phytosterol-enriched diet.
      ,
      • Moghadasian M.H.
      Dietary phytosterols reduce probucol-induced atherogenesis in apo E-KO mice.
      ,
      • Patel M.D.
      • Thompson P.D.
      Phytosterols and vascular disease.
      ]. Competition between plant sterols and intestinal cholesterol for incorporation into mixed micelles has been proposed as the mechanism undelying the hypocholesterolemic effect of plant sterols [
      • Patel M.D.
      • Thompson P.D.
      Phytosterols and vascular disease.
      ]. However, phytosterols do not need to be present in the intestinal lumen simultaneously with cholesterol to inhibit its absorption [
      • Plat J.
      • van Onselen E.N.
      • van Heugten M.M.
      • et al.
      Effects on serum lipids, lipoproteins and fat soluble antioxidant concentrations of consumption frequency of margarines and shortenings enriched with plant stanol esters.
      ]. Induction of Liver X Receptor (LXR) increases transcription of several members of the ABC gene family such as ABCA1 and ABCG5/ABCG8 [
      • Kaneko E.
      • Matsuda M.
      • Yamada Y.
      • et al.
      Induction of intestinal ATP-binding cassette transporters by a phytosterol-derived liver X receptor agonist.
      ]. Therefore, LXR induction could explain the hypocholesterolemic action of plant sterols and stanols [
      • Kaneko E.
      • Matsuda M.
      • Yamada Y.
      • et al.
      Induction of intestinal ATP-binding cassette transporters by a phytosterol-derived liver X receptor agonist.
      ]. We and others have reported that dietary plant stanols and sterols decrease intestinal cholesterol absorption independently of changes in gene expression of intestinal ABCA1, ABCG5/G8 and Niemann–Pick C1-Like 1 protein (NPC1L1) [
      • Calpe-Berdiel L.
      • Escola-Gil J.C.
      • Ribas V.
      • et al.
      Changes in intestinal and liver global gene expression in response to a phytosterol-enriched diet.
      ,
      • Field F.J.
      • Born E.
      • Mathur S.N.
      Stanol esters decrease plasma cholesterol independently of intestinal ABC sterol transporters and Niemann–Pick C1-like 1 protein gene expression.
      ]. However, these observations do not rule out a post-transcriptional action on these genes. With the creation of specific genetically engineered mice that overexpress or are deficient in these sterol transporters, a solid body of information has emerged on the mechanisms involving intestinal cholesterol absorption. This is the case of NPC1L1-deficient mice which have permitted the identification of an ezetimibe-sensitive pathway that involves intestinal NPC1L1 [
      • Altmann S.W.
      • Davis Jr., H.R.
      • Zhu L.J.
      • et al.
      Niemann–Pick C1 Like 1 protein is critical for intestinal cholesterol absorption.
      ]. The use of ABCA1-deficient mice permitted us to demonstrate that the phytosterol-mediated inhibition of intestinal cholesterol absorption is independent of this transporter [
      • Calpe-Berdiel L.
      • Escola-Gil J.C.
      • Blanco-Vaca F.
      Phytosterol-mediated inhibition of intestinal cholesterol absorption is independent of ATP-binding cassette transporter A1.
      ]. A recent report showed that plant sterol- and stanol-induced reduction in cholesterol absorption is not influenced by ABCG5-deficiency in mice [
      • Plosch T.
      • Kruit J.K.
      • Bloks V.W.
      • et al.
      Reduction of cholesterol absorption by dietary plant sterols and stanols in mice is independent of the abcg5/8 transporter.
      ]. We gained further insight into this topic via a similar, yet slightly different approach: the use of the fecal dual-isotope ratio method in ABCG5/G8 double knockout mice (Jackson Laboratories, Bar Harbor, ME, #004670) fed a 2% phytosterol-enriched Western-type diet (w/w), as previously reported [
      • Calpe-Berdiel L.
      • Escola-Gil J.C.
      • Ribas V.
      • et al.
      Changes in intestinal and liver global gene expression in response to a phytosterol-enriched diet.
      ]. Our results, in line with previous findings [
      • Calpe-Berdiel L.
      • Escola-Gil J.C.
      • Ribas V.
      • et al.
      Changes in intestinal and liver global gene expression in response to a phytosterol-enriched diet.
      ,
      • Field F.J.
      • Born E.
      • Mathur S.N.
      Stanol esters decrease plasma cholesterol independently of intestinal ABC sterol transporters and Niemann–Pick C1-like 1 protein gene expression.
      ,
      • Plosch T.
      • Kruit J.K.
      • Bloks V.W.
      • et al.
      Reduction of cholesterol absorption by dietary plant sterols and stanols in mice is independent of the abcg5/8 transporter.
      ], demonstrate that the complete absence of ABCG5/G8 heterodimer in mice does not modify the reduction in intestinal cholesterol absorption mediated by plant sterols (Table 1). We also found no major compensatory change in the intestinal gene expression of other sterol transporters such as ABCA1 or NPC1L1 (Table 1). These results provide additional evidence that the ABCG5/G8 transporter is not the molecular target of plant sterols/stanols. It remains to be elucidated whether other LXR-regulated genes or NCP1L1 could be involved in the action of plant sterols/stanols. The fact that ABCG5/G8 is required for LXR-induced reduction in intestinal cholesterol absorption [
      • Yu L.
      • York J.
      • von Bergmann K.
      • et al.
      Stimulation of cholesterol excretion by the liver X receptor agonist requires ATP-binding cassette transporters G5 and G8.
      ,
      • Plosch T.
      • Bloks V.W.
      • Terasawa Y.
      • et al.
      Sitosterolemia in ABC-transporter G5-deficient mice is aggravated on activation of the liver-X receptor.
      ] suggests that plant sterols/stanols do not act through an LXR-sensitive pathway. However, this question should be directly addressed by using LXR-deficient animals fed plant sterols and stanols. The same approach may also address the potential role of NPC1L1 in this process.
      Table 1Effects of phytosterols (2% in Western-type diet, w/w) on intestinal cholesterol absorption and relative intestinal mRNA levels of selected genes in ABCG5/G8-deficient (−/−) and ABCG5/G8 +/+ mice
      ABCG5/G8 +/+ ABCG5/G8 −/−
      Control 2% Phytosterols Control 2% Phytosterols
      Intestinal cholesterol absorption (%) 51.5 ± 4.1 24.8 ± 1.9
      P<0.05, effect of phytosterol treatment.
      56.4 ± 4.3 31.2 ± 6.2
      P<0.05, effect of phytosterol treatment.
      Relative level of ABCA1 mRNA 99.2 ± 12.0 100 ± 4.1 83.4 ± 2.6 95.8 ± 10.9
      Relative level of NPC1L1 mRNA 84.1 ± 4.5 94.9 ± 3.2 86.9 ± 4.9 100 ± 9.7
      Relative level of ABCG5 mRNA 91.7 ± 9.4 100 ± 4.2 15.6 ± 0.9
      P<0.05, effect of genotype.
      19.1 ± 0.8
      P<0.05, effect of genotype.
      Relative level of ABCG8 mRNA 98.1 ± 7.8 100 ± 3.3 7.8 ± 0.8
      P<0.05, effect of genotype.
      9.5 ± 0.4
      P<0.05, effect of genotype.
      In mRNA expression, the most abundant signal was set at a normalized value of 100 arbitrary units. Results are expressed as mean ± S.E.M. of individual animals (n = 4–5). In ABCG5/G8 −/− mice, residual ABCG5 and ABCG8 mRNA were detectable owing to the fact that PCR primers were located outside the disrupted exons, as previously reported
      • Calpe-Berdiel L.
      • Escola-Gil J.C.
      • Blanco-Vaca F.
      Phytosterol-mediated inhibition of intestinal cholesterol absorption is independent of ATP-binding cassette transporter A1.
      ,
      • Yu L.
      • York J.
      • von Bergmann K.
      • et al.
      Stimulation of cholesterol excretion by the liver X receptor agonist requires ATP-binding cassette transporters G5 and G8.
      .
      a P < 0.05, effect of phytosterol treatment.
      b P < 0.05, effect of genotype.

      Keywords

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