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A mouse model of sitosterolemia: absence of Abcg8/sterolin-2 results in failure to secrete biliary cholesterol.

Klett EL, Lu K, Kosters A, Vink E, Lee MH, Altenburg M, Shefer S, Batta AK, Yu H, Chen J, Klein R, Looije N, Oude-Elferink R, Groen AK, Maeda N, Salen G, Patel SB - BMC Med (2004)

Bottom Line: No phenotypic difference has yet been described in humans with complete defects in either ABCG5 or ABCG8.These proteins, based upon the defects in humans, are responsible for regulating dietary sterol entry and biliary sterol secretion.These data indicate that Abcg8/sterolin-2 is necessary for biliary sterol secretion and that loss of Abcg8/sterolin-2 has a more profound effect upon biliary cholesterol secretion than sitosterol.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Endocrinology, Diabetes and Medical Genetics, Medical University of South Carolina, Charleston, SC 29403, USA. klettel@musc.edu

ABSTRACT

Background: Mutations in either of two genes comprising the STSL locus, ATP-binding cassette (ABC)-transporters ABCG5 (encoding sterolin-1) and ABCG8 (encoding sterolin-2), result in sitosterolemia, a rare autosomal recessive disorder of sterol trafficking characterized by increased plasma plant sterol levels. Based upon the genetics of sitosterolemia, ABCG5/sterolin-1 and ABCG8/sterolin-2 are hypothesized to function as obligate heterodimers. No phenotypic difference has yet been described in humans with complete defects in either ABCG5 or ABCG8. These proteins, based upon the defects in humans, are responsible for regulating dietary sterol entry and biliary sterol secretion.

Methods: In order to mimic the human disease, we created, by a targeted disruption, a mouse model of sitosterolemia resulting in Abcg8/sterolin-2 deficiency alone. Homozygous knockout mice are viable and exhibit sitosterolemia.

Results: Mice deficient in Abcg8 have significantly increased plasma and tissue plant sterol levels (sitosterol and campesterol) consistent with sitosterolemia. Interestingly, Abcg5/sterolin-1 was expressed in both liver and intestine in Abcg8/sterolin-2 deficient mice and continued to show an apical expression. Remarkably, Abcg8 deficient mice had an impaired ability to secrete cholesterol into bile, but still maintained the ability to secrete sitosterol. We also report an intermediate phenotype in the heterozygous Abcg8+/- mice that are not sitosterolemic, but have a decreased level of biliary sterol secretion relative to wild-type mice.

Conclusion: These data indicate that Abcg8/sterolin-2 is necessary for biliary sterol secretion and that loss of Abcg8/sterolin-2 has a more profound effect upon biliary cholesterol secretion than sitosterol. Since biliary sitosterol secretion is preserved, although not elevated in the sitosterolemic mice, this observation suggests that mechanisms other than by Abcg8/sterolin-2 may be responsible for its secretion into bile.

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Generation of mice deficient in Abcg8/sterolin-2. The targeted disruption strategy of Abcg8 is as shown (panel a). Southern blot analysis of BamHI digested mouse genomic DNA, probed with [32P]-randomly labelled probe resulting in a 6.0 kb band for wild type, a 2.7 kb band for homozygous and two bands of 5.9 and 2.6 kb for the heterozygote (panel b). Northern blot analysis of hepatic RNA showed a loss of Abcg8/sterolin-2 mRNA in the homozygote and decreased Abcg8/sterolin-2 mRNA in the heterozygote, although Abcg5/sterolin-1 mRNA appeared relatively unaffected in the knockout mice (panel c). Probes were approximately 1.9 kb for Abcg5 and 2 kb for Abcg8 (see Methods for more detail). RT-PCR analyses of hepatic cDNA showed no Abcg8/sterolin-2 message, downstream of exon 4 in the Abcg8-/- mice, whether primers were located in exons 4 and 13 (panel d), exons 9 and 13 or exons 10 and 13 (panel e).
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Figure 1: Generation of mice deficient in Abcg8/sterolin-2. The targeted disruption strategy of Abcg8 is as shown (panel a). Southern blot analysis of BamHI digested mouse genomic DNA, probed with [32P]-randomly labelled probe resulting in a 6.0 kb band for wild type, a 2.7 kb band for homozygous and two bands of 5.9 and 2.6 kb for the heterozygote (panel b). Northern blot analysis of hepatic RNA showed a loss of Abcg8/sterolin-2 mRNA in the homozygote and decreased Abcg8/sterolin-2 mRNA in the heterozygote, although Abcg5/sterolin-1 mRNA appeared relatively unaffected in the knockout mice (panel c). Probes were approximately 1.9 kb for Abcg5 and 2 kb for Abcg8 (see Methods for more detail). RT-PCR analyses of hepatic cDNA showed no Abcg8/sterolin-2 message, downstream of exon 4 in the Abcg8-/- mice, whether primers were located in exons 4 and 13 (panel d), exons 9 and 13 or exons 10 and 13 (panel e).

Mentions: A mouse genomic bacterial artificial chromosome (BAC) library (CitbCJ7, ES cell line/129Sv, Research Genetics, Inc., Huntsville, AL, USA) was screened by using primers designed from the sequences of mouse Abcg5 and Abcg8 cDNA as previously reported [21]. A positive BAC clone was used as a template to amplify genomic DNA fragments of Abcg8. Long-fragment polymerase chain reaction (PCR) was performed using Expanded Long Template PCR system kit (Roche Applied Science, Indianapolis, IA, USA). An approximately 4.5 kb 'long-arm' genomic fragment containing partial exon 1 to partial exon 3 was inserted into the Pml I restriction-cloning site A of OSDUPDEL vector. The 'short-arm' genomic fragment, containing partial exon 4 to partial exon 6, was cloned into the Not I-Kpn I of cloning site B. Homologous targeting would result in complete intron 3, partial exon 3 and partial exon 4 replacement by the neomycin-resistance cassette, resulting in the disruption of the ABC Walker A motif (Figure 1a).


A mouse model of sitosterolemia: absence of Abcg8/sterolin-2 results in failure to secrete biliary cholesterol.

Klett EL, Lu K, Kosters A, Vink E, Lee MH, Altenburg M, Shefer S, Batta AK, Yu H, Chen J, Klein R, Looije N, Oude-Elferink R, Groen AK, Maeda N, Salen G, Patel SB - BMC Med (2004)

Generation of mice deficient in Abcg8/sterolin-2. The targeted disruption strategy of Abcg8 is as shown (panel a). Southern blot analysis of BamHI digested mouse genomic DNA, probed with [32P]-randomly labelled probe resulting in a 6.0 kb band for wild type, a 2.7 kb band for homozygous and two bands of 5.9 and 2.6 kb for the heterozygote (panel b). Northern blot analysis of hepatic RNA showed a loss of Abcg8/sterolin-2 mRNA in the homozygote and decreased Abcg8/sterolin-2 mRNA in the heterozygote, although Abcg5/sterolin-1 mRNA appeared relatively unaffected in the knockout mice (panel c). Probes were approximately 1.9 kb for Abcg5 and 2 kb for Abcg8 (see Methods for more detail). RT-PCR analyses of hepatic cDNA showed no Abcg8/sterolin-2 message, downstream of exon 4 in the Abcg8-/- mice, whether primers were located in exons 4 and 13 (panel d), exons 9 and 13 or exons 10 and 13 (panel e).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC394351&req=5

Figure 1: Generation of mice deficient in Abcg8/sterolin-2. The targeted disruption strategy of Abcg8 is as shown (panel a). Southern blot analysis of BamHI digested mouse genomic DNA, probed with [32P]-randomly labelled probe resulting in a 6.0 kb band for wild type, a 2.7 kb band for homozygous and two bands of 5.9 and 2.6 kb for the heterozygote (panel b). Northern blot analysis of hepatic RNA showed a loss of Abcg8/sterolin-2 mRNA in the homozygote and decreased Abcg8/sterolin-2 mRNA in the heterozygote, although Abcg5/sterolin-1 mRNA appeared relatively unaffected in the knockout mice (panel c). Probes were approximately 1.9 kb for Abcg5 and 2 kb for Abcg8 (see Methods for more detail). RT-PCR analyses of hepatic cDNA showed no Abcg8/sterolin-2 message, downstream of exon 4 in the Abcg8-/- mice, whether primers were located in exons 4 and 13 (panel d), exons 9 and 13 or exons 10 and 13 (panel e).
Mentions: A mouse genomic bacterial artificial chromosome (BAC) library (CitbCJ7, ES cell line/129Sv, Research Genetics, Inc., Huntsville, AL, USA) was screened by using primers designed from the sequences of mouse Abcg5 and Abcg8 cDNA as previously reported [21]. A positive BAC clone was used as a template to amplify genomic DNA fragments of Abcg8. Long-fragment polymerase chain reaction (PCR) was performed using Expanded Long Template PCR system kit (Roche Applied Science, Indianapolis, IA, USA). An approximately 4.5 kb 'long-arm' genomic fragment containing partial exon 1 to partial exon 3 was inserted into the Pml I restriction-cloning site A of OSDUPDEL vector. The 'short-arm' genomic fragment, containing partial exon 4 to partial exon 6, was cloned into the Not I-Kpn I of cloning site B. Homologous targeting would result in complete intron 3, partial exon 3 and partial exon 4 replacement by the neomycin-resistance cassette, resulting in the disruption of the ABC Walker A motif (Figure 1a).

Bottom Line: No phenotypic difference has yet been described in humans with complete defects in either ABCG5 or ABCG8.These proteins, based upon the defects in humans, are responsible for regulating dietary sterol entry and biliary sterol secretion.These data indicate that Abcg8/sterolin-2 is necessary for biliary sterol secretion and that loss of Abcg8/sterolin-2 has a more profound effect upon biliary cholesterol secretion than sitosterol.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Endocrinology, Diabetes and Medical Genetics, Medical University of South Carolina, Charleston, SC 29403, USA. klettel@musc.edu

ABSTRACT

Background: Mutations in either of two genes comprising the STSL locus, ATP-binding cassette (ABC)-transporters ABCG5 (encoding sterolin-1) and ABCG8 (encoding sterolin-2), result in sitosterolemia, a rare autosomal recessive disorder of sterol trafficking characterized by increased plasma plant sterol levels. Based upon the genetics of sitosterolemia, ABCG5/sterolin-1 and ABCG8/sterolin-2 are hypothesized to function as obligate heterodimers. No phenotypic difference has yet been described in humans with complete defects in either ABCG5 or ABCG8. These proteins, based upon the defects in humans, are responsible for regulating dietary sterol entry and biliary sterol secretion.

Methods: In order to mimic the human disease, we created, by a targeted disruption, a mouse model of sitosterolemia resulting in Abcg8/sterolin-2 deficiency alone. Homozygous knockout mice are viable and exhibit sitosterolemia.

Results: Mice deficient in Abcg8 have significantly increased plasma and tissue plant sterol levels (sitosterol and campesterol) consistent with sitosterolemia. Interestingly, Abcg5/sterolin-1 was expressed in both liver and intestine in Abcg8/sterolin-2 deficient mice and continued to show an apical expression. Remarkably, Abcg8 deficient mice had an impaired ability to secrete cholesterol into bile, but still maintained the ability to secrete sitosterol. We also report an intermediate phenotype in the heterozygous Abcg8+/- mice that are not sitosterolemic, but have a decreased level of biliary sterol secretion relative to wild-type mice.

Conclusion: These data indicate that Abcg8/sterolin-2 is necessary for biliary sterol secretion and that loss of Abcg8/sterolin-2 has a more profound effect upon biliary cholesterol secretion than sitosterol. Since biliary sitosterol secretion is preserved, although not elevated in the sitosterolemic mice, this observation suggests that mechanisms other than by Abcg8/sterolin-2 may be responsible for its secretion into bile.

Show MeSH
Related in: MedlinePlus