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].