Results

Isolation of Abcg8/sterolin-2 deficient mice
The targeting construct (Figure 1a) results in the potential disruption of normal splicing, as well as loss of some coding sequences involving exons 3 and 4. After ES cell electroporation with the linearized targeted plasmid DNA, two homologous recombinant ES clones (out of 58 ES clones screened) were identified. Blastocyst injection of these resulted in five highly chimeric mice (three males, two females). One male and one female, both from the same ES clone, showed germline transmission and the female line was used to establish a multi-generational colony bred onto a C57Bl/6J background.
Heterozygous mice were fertile and gave rise to normal litter sizes. Breeding heterozygous animals led to wild-type, heterozygous and homozygous knockout mice in the expected Mendelian distribution (data not shown). Although not quantitative, Northern analyses showed that while the knockout mice showed no detectable mRNA for Abcg8, expression of Abcg5 appeared unaltered (Figure 1c). To exclude the possibility of alternative splicing with the production of a non-functional but truncated Abcg8/sterolin-2 protein that may serve as a chaperone for Abcg5/sterolin-1, RT-PCR was performed on cDNA reverse transcribed from total liver RNA from Abcg8-deficient mice. No mRNA for Abcg8/sterolin-2 was detected containing any sequences downstream of exon 4 in the Abcg8-deficient mice, whether primers were located in exons 4 and 13, exons 9 and 13 or exons 10 and 13 (Figure 1d,1e). This demonstrates that if an alternatively spliced message could potentially code for a truncated protein, it is not detectable in the Abcg8-deficient mice. The membrane-spanning domains of Abcg8/sterolin-2 are encoded by exons 9–13.

Sterol levels in Abcg8/sterolin-2 deficient animals
Elevated plant sterols in tissue and plasma are diagnostic of sitosterolemia. Sterol levels, as determined by gas chromatography (GC) analysis, in plasma and tissues of the Abcg8-/- mice are shown in Table 2. There were no differences in tissue weights between wild types, heterozygotes and knockouts. Plasma cholesterol levels of the homozygous and heterozygous mice were decreased by 52% and 26%, respectively, compared to those of wild-type mice (n = 3 all groups, Table 2). Plant sterols were almost undetectable in wild-type and heterozygous mice, but were significantly elevated in Abcg8-/- mice. Plasma campesterol and sitosterol levels were eight- and 36-fold higher in Abcg8-/- mice compared to wild-type mice (8.76 ± 2.10 and 18.52 ± 5.03 mg/dL, respectively, versus 1.14 ± 0.69 and 0.50 ± 0.55 mg/dL). There was no significant difference in the plasma plant sterol levels between heterozygous and wild-type mice. Liver cholesterol content of the Abcg8-/- mice was reduced by ~50% relative to wild-type liver (1202 ± 264 μg/g wet weight tissue, versus 2280 ± 310 μg/g wet weight tissue, respectively). The reduced cholesterol content was offset by a five-fold increase in campesterol and a 22-fold increase in sitosterol (233.9 ± 48.1 and 376.3 ± 96.1 μg/g wet weight tissue, respectively) in Abcg8-/- knockout mice compared to wild-type mice (52.2 ± 15.0 and 16.7 ± 8.6 μg/g wet weight tissue, respectively, Table 2). There were no significant differences of liver cholesterol or plant sterol contents between Abcg8+/+ and Abcg8+/- mice. Spleen sterol contents reflected the liver profiles (Table 2). No significant differences in brain sterol contents were observed, as would be expected, since the blood brain barrier is intact and prevents entry of these sterols in sitosterolemia (Table 2). There were only traces of plant sterols in the brains from knockout animals, reflecting blood contamination during tissue harvesting.
Interestingly, the majority of the elevated tissue plant sterols are unesterified. The livers of Abcg8+/+, +/- and -/- mice have relatively similar levels of esterified cholesterol (Figure 2a). However, there is little if any esterification of sitosterol or campesterol (Figure 2b,2c) consistent with previous findings [37,38]. Thus all of the expansion of tissue sterol pools are as free sterols.
FPLC analyses for sterols (measured enzymatically and thus reflecting total sterols) and triglycerides were performed on plasma samples from fasted animals fed a regular chow diet. No significant differences were observed for the sterol profiles (Figure 3a), but surprisingly, the Abcg8-/- knockout mice had a significantly higher triglyceride level in the LDL lipoprotein fractions compared to wild-type and heterozygous mice (Figure 3b). The size of this peak was variable between different littermate analyses, but is always increased in the knockout animals. Total plasma triglyceride levels of the Abcg8-/- mice were slightly higher (79.2 ± 14 mg/dL compared to the wild type 63.4 ± 13 mg/dL and heterozygotes 46.6 ± 12 mg/dL, n = 3 for all genotypes). Preliminary analyses of proteins in the isolated FPLC fractions did not show any qualitative changes in apolipoproteins B, E, AI, or AII. The significance of this triglyceride rich peak remains unclear at present.

Liver gene expression and enzyme activity change in Abcg8/sterolin-2 deficient mice
To investigate the effects of a deficiency of Abcg8/sterolin-2 on the genes that regulate sterol metabolism, quantitative RT-PCR was performed looking at the expression levels of Abcg5, Abcg8, Hmgr, Cyp7a1, Abca1, Mdr2, Lxr, Srebp-1c, and Srebp-2 mRNA in the livers of mice fed a regular chow diet (Figure 4a). As expected, Abcg8 mRNA expression levels were undetectable in the Abcg8-/- mice and were reduced by ~50% in the heterozygous mice, relative to wild-type mice. Interestingly, by quantitative RT-PCR, the mRNA expression of Abcg5 in the knock-out mice was also reduced by more than 60% compared to the wild-type mice, although no changes were noted in the heterozygous mice. Expression of HMG-CoA reductase mRNA was decreased by ~50% and ~80% in the heterozygote and knockout mice respectively, in keeping with limited observations in human patients with this disorder [7].
To verify whether the mRNA changes resulted in alteration of the enzyme activity changes, liver samples were analyzed for HMG-CoA reductase activity and Cyp7a1 activity (Figure 4b). HMG-CoA reductase activity was reduced by 30% and 60% in the Abcg8+/- and Abcg8-/- mice, respectively (Figure 4b), and thus reflected the changes in mRNA expression. In contrast, although the Cyp7a1 mRNA expression levels were essentially unchanged in the knockout mouse, Cyp7a1 activity was significantly decreased by 37% (P < 0.01). In the heterozygous mice, both the mRNA and activity of Cyp7a1 were decreased. Sitosterol is known to be a direct competitive inhibitor of Cyp7a1 and it is likely that the elevated plant sterols in the liver are responsible for the inhibition in the knockout mouse [39].

Localization of Abcg5 protein in Abcg8-/- knockout mice
Does the loss of Abcg8/sterolin-2 result in loss of Abcg5/sterolin-1 expression, as might be predicted from the genetic studies and more recently from the in vitro and in vivo expression studies? A robust antibody to mouse Abcg8/sterolin-2 is not currently available. However, three separate groups have developed rabbit polyclonal antibody to mouse Abcg5/sterolin-1. These three antibodies were used in Western blotting and immunohistochemistry experiments on the liver and intestine of Abcg8-/- mice. Western blotting of liver total membrane preparations using the three separately-developed anti-Abcg5/sterolin-1 antibodies showed different results. As has been previously published, Abcg5/sterolin-1 exists in two separate forms, the 'immature' 75 kDa protein and a fully glycosylated 'mature' 93 kDa protein [18,30]. Using the SC anti-Abcg5/sterolin-1 peptide antibody, a 75 kDa band is detected (Figure 5a). This antibody does not detect a 'mature' 93 kDa band in either wild-type or knockout animals nor is the 75 kDa band sensitive to either Endo-H or PNGaseF (lower panel shows same aliquots probed with anti-transferrin). The AMC antibody detects the 'mature' form of Abcg5/sterolin-1 in wild-type animals but not the knockouts. Interestingly, the 'immature' 75 kDa band is detected by this antibody but the band shifts with Endo-H and PNGase F treatment (Figure 5b). The UTSW antibody detects the 'mature' and 'immature' forms of Abcg5/sterolin-1 in wild-type animals but detects no forms in the knockout mouse (Figure 5c). What does hold true for all antibodies used against Abcg5/sterolin-1 is the detection of a 75 kDa band.
Immunohistochemistry experiments were carried out using all three antibodies. In acute transfection studies, the SC antibody recognized over-expressed mouse Abcg5/sterolin-1 in COS-1 cells, but not mouse Abcg8/sterolin-2 (Figure 6b,6c). Co-transfection of COS-1 cells with both Abcg5 and Abcg8 cDNAs did not alter the pattern of immunofluorescence, although we are not able to confirm simultaneous expression of Abcg8 in cells expressing Abcg5/sterolin-1, as we have no suitable antibody for Abcg8/sterolin-2 (Figure 6d). Nevertheless, this experiment indicates that the SC antibody can recognize Abcg5/sterolin-1 but does not cross-react with Abcg8/sterolin-2.
Serial sections of intestinal tissue were incubated with anti-Abcg5/sterolin-1 to determine the cellular location of Abcg5/sterolin-1. The pattern of staining of Abcg5/sterolin-1 in the intestinal sections is clearly apical and localized to the villi of the enterocytes (Figure 6g,6h,6i). Loss of Abcg8/sterolin-2 does not seem to affect this pattern of staining. To further confirm that the antibody used recognizes Abcg5/sterolin-1; this pattern is blocked if the antibody is pre-incubated with the peptide to which it was raised (Figure 6f). To further confirm that Abcg5/sterolin-1 expression is preserved and may be apical, immunohistochemistry was performed in Amsterdam using the AMC antibody to stain liver sections from wild-type and knockout mice [30]. As a control, the expression of Abcb11, an ABC transporter known to be responsible for the export of bile salts, was compared to that of Abcg5/sterolin-1 (Figure 6j,6k). Again, both proteins are apically localized in liver sections from wild-type mice and this pattern is essentially unperturbed in liver from knockout mice, although the expression level of Abcg5/sterolin-1 seems to be less robust qualitatively (Figure 6l,6m). To further confirm the apical expression of Abcg5/sterolin-1 in the knockout mice, the UTSW antibody was used to stain liver sections of wild-type and knockout mice. Again, there is apical expression of Abcg5/sterolin-1 in both wild-type and knockout livers (Figure 7a,7b).

Biliary secretion of bile salts, sterol and phospholipids in Abcg8/sterolin-2 deficient mice
Sitosterolemic individuals have an impaired ability to secrete cholesterol and plant sterols into bile [40,41]. We analyzed the knockout mice for any alterations in biliary sterol handling. Analyses of the initial basal bile secretion showed that the sterol and phospholipid contents in Abcg8-/- mice were reduced as compared to wild-type mice, (Figure 8a,8b,8c, bile salt secretion not statistically significant, for sterol secretion P = 0.01 and for phospholipid secretion P = 0.03).
To investigate whether the defect in biliary sterol secretion could be (partly) restored by forced biliary sterol secretion, we infused mice with stepwise increasing doses of tauroursodeoxycholic acid (TUDC). Mice were first depleted of their endogenous bile salt pools for 90 min and subsequently infused via the jugular vein with increasing doses of TUDC. As shown in Figure 8d, bile salt secretion was no different between the different genotypes during the depletion or the TUDC infusion phases of the experiment. However, a different pattern emerged for the secretion of phospholipid (Figure 8f). There was no difference between wild-type and heterozygous mice, but knockout mice showed a trend of lower phospholipid secretion during both the depletion and infusion phase. Even more dramatic was the effect on biliary sterol secretion although the sterol contents were determined enzymatically and no distinction is made as to whether this is sitosterol or cholesterol (Figure 8e, but see below). In the knockout mice, almost no stimulated sterol secretion was noted, with an intermediate phenotype in the heterozygous mice. In the heterozygous mice, sterol secretion increased to reach a maximal level of about 50% of the wild type suggesting that Abcg8/sterolin-2 is a rate-limiting step for biliary 'cholesterol' secretion. In the knockout mice, sterol secretion remained at a constant level during the depletion phase and increased minimally upon infusion of TUDC.
Since the Abcg8-/- mice still secreted some sterols we were interested in which biliary sterol species were being secreted. Therefore bile was collected from mice during a constant infusion of TUDC and analyzed by GC. Mice were first depleted of their endogenous bile acid pools for 30 minutes then infused with a continuous dose of TUDC (1,200 nmol/min/100 g body weight). The knockout mice showed a significantly diminished ability to secrete cholesterol, yet still maintained the ability to secrete sitosterol and campesterol compared to wild-type mice (Figure 9). Surprisingly, the Abcg8+/- mice tended to show an increased ability to secrete all sterols above the levels seen in the wild-type mice, although this was not statistically significant.