Figures and Tables
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).
Figure 2  Free versus esterified sterol levels in the livers of Abcg8+/+, Abcg8+/- and Abcg8-/- mice. Sterol contents of liver extracts of 12-week-old female mice fed a regular chow diet, determined by GC analysis show that the majority of sterols in all genotypes are unesterified. Esterified cholesterol (panel a) remains relatively constant for each genotype. However, no esterified sitosterol in the livers from Abcg8+/+ and Abcg8+/- mice and very little from Abcg8-/- mice was detected (panel b). Small amounts of esterified campesterol were detected in each of the genotypes (panel c).
Figure 3  Lipid profiles of the plasma of Abcg8+/+, Abcg8+/- and Abcg8-/- mice. Lipoproteins were separated from pooled mouse plasma samples by FPLC. Total sterols (panel a) and triglyceride (panel b) profiles of the fractions are shown. There was no difference of cholesterol profiles in the groups, but there was a small triglyceride peak in Abcg8-/- mice in fractions 21–25, corresponding to the LDL-size range, the significance of which is not known at present.
Figure 4  Analyses of mRNA expression and enzyme activity in mouse livers. Panel (a) shows RT-PCR quantitation of mRNA levels for Abcg5, Abcg8, Hmgr, Cyp7a1, Abca1, Mdr2, Lxr, Srebp-1c and Srebp-2 in mouse livers from Abcg8+/+ (open bars), Abcg8+/- (hatched bars) and Abcg8-/- (filled bars). Knockout mice showed an ~60% reduction in mRNA for Abcg5/sterolin-1 and an 80% reduction in the message for HMG CoA reductase. Relatively no message for Abcg8/sterolin-2 was detected in the knockout mice. Panel (b) shows the enzyme activities for HMG-CoA reductase and CYP7a1 in livers from Abcg8+/+, Abcg8+/- and Abcg8-/- mice. Activities of HMG-CoA reductase and CYP7a1 were significantly reduced in the Abcg8-/- mouse liver (*P < 0.05, see text for discussion).
Figure 5  N-glycosylation of Abcg5/sterolin-1 analyses in Abcg8-/- mouse liver. Mouse liver homogenate stained with SC anti-Abcg5/sterolin-1 after treatment with Endo-H or PNGaseF shows a 75 kDa band present in both genotypes, which is resistant to deglycosylation (panel a). Staining of wild-type liver homogenate with preimmune serum showed no detectable bands. Lower portion of panel (a) shows the same aliquots stained for anti-transferrin as a control for deglycosylation. AMC anti-Abcg5/sterolin-1 staining of mouse liver homogenate shows a 'mature' ~90 kDa band in the wild-type mice but not in the Abcg8-/- mice (panel b). A 75 kDa form is present which is sensitive to deglycosylation. Mouse liver homogenate stained with UTSW anti-Abcg5/sterolin-1 shows a 'mature' ~90 kDa band and an 'immature' 75 kDa band in the wild-type mice but no signal is detected in the Abcg8-/- mice. Treatment with Endo-H or PNGaseF results in a lower molecular weight protein in the wild-type mice. Abcg5/Abcg8-/- liver homogenate used for negative control.
Figure 6  Immunohistochemical evaluation of mouse Abcg5/sterolin-1 expression in liver and intestine. COS-1 cells were transiently transfected with pCMV-mouse Abcg5 or pCMV-mouse Abcg8 constructs, allowed to express for 48 hours then fixed and incubated with antibody. pCMV empty vector transfected COS-1 cells incubated with SC anti-Abcg5/sterolin-1 showed no significant fluorescence (panel a). Abcg5 transfected COS-1 cells incubated with SC anti-Abcg5/sterolin-1 antibody showed a membrane distribution (panel b), where as Abcg8 transfected cells incubated with SC anti-Abcg5 antibody showed no significant fluorescence (panel c). Abcg5 and Abcg8 co-transfected COS-1 cells incubated with SC anti-Abcg5/sterolin-1 antibody resulted in a fluorescence pattern similar to Abcg5 alone (panel d). The yellow bar represents 20 μm. Wild-type intestine incubated with SC pre-immune serum (panel e), or SC anti-Abcg5/sterolin-1 antibody pre-incubated with the blocking peptide (panel f) showed no specific signals. Wild-type, Abcg8+/- and Abcg8-/- intestine (panels g, h and i respectively) incubated with SC anti-Abcg5/sterolin-1 antibody, showed no difference in expression patterns, despite the loss of Abcg8/sterolin-2 in the knockout mice. Single arrowhead shows intestinal villus and double arrowhead shows crypt. The yellow bar represents 50 μm. Antibody staining of liver sections was also performed at the AMC Liver Center. As a control, an antibody to Bsep/Abcb11 was used and showed a clear apical distribution in both wild-type (panel j) and Abcg8 knockout mice (panel k). Using AMC antibody against Abcg5/sterolin-1 (see text), in both wild-type (panel l) and Abcg8 knockout liver (panel m), the pattern of expression was also apical and unchanged although the signal is fainter compared to that for Bsep/Abcb11 (see text for discussion). Arrows indicate bile canaliculi.
Figure 7  Immunohistochemical evaluation of mouse Abcg5/sterolin-1 expression in liver. Wild-type liver incubated with UTSW anti-Abcg5/sterolin-1 resulted in apical expression (panel a). Merged image clearly shows the apical distribution. Likewise Abcg8-/- liver incubated with the same UTSW anti-Abcg5/sterolin-1 antibody resulted in a similar apical expression pattern relative to the wild type (panel b). White arrows indicate bile canaliculi and asterisk indicates a bile duct. The yellow bar represents 50 μm.
Figure 8  Biliary sterol, phospholipid and bile salt analyses. Bile salt, sterol and phospholipid contents from male Abcg8+/+ (n = 8), Abcg8+/- (n = 7), and Abcg8-/- (n = 5) mice were examined as described in Methods. Bile salt secretion following a 10 minute collection was lower in Abcg8+/- and Abcg8-/- mice compared to wild type (panel a), but these differences were not statistically significant. Biliary sterol and phospholipid were significantly reduced in the Abcg8-/- mice compared to the wild-type (panels b and c). *P < 0.05. The lower panel shows biliary bile salt, sterol and phospholipid secretion rates from female Abcg8+/+ (n = 5), Abcg8+/- (n = 4), and Abcg8-/- (n = 3) mice measured following 90-minute bile salt depletion followed by stepwise TUDC infusion as described in the text. Bars represent phase TUDC infusion rates. No differences were observed in the ability of the Abcg8-/- mice to secrete bile salts (panel d), but there is a marked inability of the knockout mice to secrete sterols (panel e) and a trend towards a reduced ability to secrete phospholipid (panel f) compared to wild type.
Figure 9  Biliary cholesterol and sitosterol secretion. Biliary sterols were analyzed by GC analyses in Abcg8+/+ (n = 4), Abcg8+/- (n = 3) and Abcg8-/- (n = 4) mice following 30-minute bile salt depletion followed by a continuous TUDC infusion as described in Methods. Abcg8-/- mice were unable to secrete cholesterol into bile with forced TUDC administration relative to the wild-type mice (panel a, *P < 0.05). Interestingly, the Abcg8-/- mice were able to still secrete sitosterol (panel b) and campesterol (panel c). Heterozygous mice showed an increased ability to secrete all sterols with forced TUDC administration, although the results were not statistically significant.
Table 1  Oligonucleotide primers used for quantitative RT-PCR
Target gene  Primer sequence  Length of amplicon  GenBank accession no.
Abcg5  Forward: 5'-AGGTCATGATGCTAGATGAGC-3'  260 bp  AH011511
Reverse: 5'-CAAAGGGATTGGAATGTTCAG-3'
Abcg8  Forward: 5'-CTCCTCGGAAAGTGACAACAG-3'  190 bp  AH011518
Reverse: 5'-TAGATTTCGGATGCCCAGCTC-3'
Hmgr  Forward: 5'-CCGGCAACAACAAGATCTGTG-3'  114 bp  BB664708
Reverse: 5'-ATGTACAGGATGGCGATGCA-3'
Cyp7a1  Forward: 5'-CAGGGAGATGCTCTGTGTTCA-3'  121 bp  NM_007824
Reverse: 5'-AGGCATACATCCCTTCCGTGA-3'
Abca1  Forward: 5'-CCCAGAGCAAAAAGCGACTC-3'  89 bp  NM_013454
Reverse: 5'-GGTCATCATCACTTTGGTCCTTG-3'
Mdr2 (Abcb4)  Forward: 5'-GCAGCGAGAAACGGAACAG-3'  64 bp  NM_008830
Reverse: 5'-GGTTGCTGATGCTGCCTAGTT-3'
LXRα  Forward: 5'-GCTCTGCTCATTGCCATCAG-3'  79 bp  AF085745
Reverse: 5'-TGTTGCAGCCTCTCTACTTGGA-3'
Srebp-1c  Forward: 5'-GGAGCCATGGATTGCACATT-3'  103 bp  BI656094
Reverse: 5'-CCTGTCTCACCCCCAGCATA-3'
Srebp-2  Forward: 5'-CTGCAGCCTCAAGTGCAAAG-3'  119 bp  AF374267
Reverse: 5'-CAGTGTGCCATTGGCTGTCT-3'
Cyclophilin  Forward: 5'-AAGTTCCATCGTGTCATCAAGGAC-3'  173 bp  M60456
Reverse: 5'-CCATTGGTGTCTTTGCCTGC-3'
Table 2  Mouse plasma and tissue sterol analyses
Plasma (mg/dL)
Group  Cholesterol  Campesterol  Stigmasterol  β-Sitosterol
Abcg8+/+  73.0 ± 14  1.1 ± 0.7  ND  0.5 ± 0.5
Abcg8+/-  54.0 ± 10*  1.8 ± 0.3*  ND  0.8 ± 0.2*
Abcg8-/-  35.0 ± 6.0*  8.8 ± 2.1*  ND  18.5 ± 5.0*
Liver (μg/g wet tissue)
Group  Cholesterol  Campesterol  Stigmasterol  β-Sitosterol
Abcg8+/+  2280 ± 310  52.2 ± 15.0  ND  16.7 ± 8.6
Abcg8+/-  2460 ± 588  76.4 ± 12.3*  1.6 ± 3.3*  26.7 ± 6.4*
Abcg8-/-  1202 ± 264*  233.9 ± 48.1*  22.7 ± 6.3*  376.3 ± 91.6*
Spleen (μg/g wet tissue)
Group  Cholesterol  Campesterol  Stigmasterol  β-Sitosterol
Abcg8+/+  2702 ± 310  41.7 ± 7.6  15.1 ± 14.1  21.9 ± 10.9
Abcg8+/-  2578 ± 320  66.8 ± 18.8*  14.5 ± 16.9*  32.2 ± 6.1*
Abcg8-/-  1779 ± 195*  353.8 ± 97.5*  ND*  436.6 ± 148.5*
Brain (μg/g wet tissue)
Group  Cholesterol  Plant sterols  Desmosterol  Lathosterol
Abcg8+/+  11301 ± 156  10.2 ± 3.9  102.0 ± 13.3  52.0 ± 0.3
Abcg8+/-  11846 ± 814  24.5 ± 5.5  113.7 ± 25.5  49.1 ± 4.2
Abcg8-/-  11846 ± 1061  63.0 ± 6.5  123 ± 18.3  56.9 ± 9.5
Animals used in these studies were 12 weeks of age, a mixture of male and female and fed a regular chow diet. *P < 0.05 for -/- versus +/+ and -/- versus +/-. ND, not detectable.