The mechanism by which dietary cholesterol is specifically absorbed and dietary non-cholesterol sterols primarily excluded or the mechanism(s) by which the liver can selectively secrete sterols has not been elucidated. Identification of the genetic defect(s) in a rare human disorder, sitosterolemia, where these processes are specifically disrupted, may finally have led to the identification of the 'transporters' responsible for these processes. Complete defects in one of two genes (but not both), organized in a head-to-head configuration at the STSL locus, causes sitosterolemia. We report a mouse model of sitosterolemia, with a selective, but complete, defect in one of these genes, Abcg8. This mouse reflects the known defects described in human sitosterolemia [5, 7, 42]. Plasma and all tissues, apart from the brain, have significantly elevated sitosterol levels. Homozygous knockout mice are viable, fertile and sitosterolemic. However, fertility seems to be reduced when homozygous mice are bred together (S Patel and J Oatis, unpublished observation).
This model reflects many other observed changes described in limited studies in humans. For example, in humans, the activity of HMG-CoA reductase and CYP7a1 have been reported to be low [7, 39]. In this study, we show that the mRNA and enzyme activities in the livers from knockout animals are also significantly reduced. Additionally, the activity of the rate-limiting enzyme for bile acid synthesis, CYP7a1, is reduced, although no changes at the mRNA level are noted. This is also in keeping with previous studies that show that sitosterol is a direct inhibitor of this enzyme in vitro .
Biliary sterols of Abcg8-deficient mice were dramatically different compared to wild-type mice. Measurement of biliary sterol secretion rates in Abcg8-/- mice demonstrated a failure to increase sterol secretion into bile despite exogenous infusion of bile acids. Furthermore, complete loss of Abcg8/sterolin-2 results in an inability to secrete cholesterol into bile, although secretion of sitosterol seemed to be preserved. Arguably, since the body pools and plasma sitosterol levels in the knockout mice are so considerably elevated, perhaps the biliary sitosterol levels could be considered to be inappropriately low. Despite this reservation, the finding that sitosterol is present in the bile suggests that plant sterols may be secreted independent of Abcg8/sterolin-2. The ability of Abcg8-/- mice to secrete bile salt and phospholipid into bile as compared to wild-type mice was not significantly impaired. Collectively these data suggest that Abcg8/sterolin-2 is necessary for hepatobiliary cholesterol secretion in mice.
Abcg8-/- mice appeared normal and healthy maintained on a regular rodent chow diet. Their phenotypic features are very similar to those recently reported for a mouse deficient in both sterolins simultaneously [7, 20]. Plasma and hepatic plant sterol levels in Abcg8-/- and Abcg5/Abcg8 knockout are increased similarly with marked decreases in cholesterol levels. Interestingly, plasma triglyceride levels of the Abcg8-/- mice were slightly higher than wild-type animals and this increased triglyceride is carried in the LDL fraction range, as measured by FPLC. The significance of this is not clear, although preliminary SDS-PAGE analyses of all the fractions failed to reveal any differences between wild-type and knockout samples.
Hepatic levels of GC-measured cholesterol were greatly reduced in Abcg8-/- mice compared to wild-type mice, yet mRNA levels of HMG-CoA reductase and enzyme activity are also reduced without any significant change in mRNA of the Srebps. The basis for this is not clear at present, unless the increase in non-plant sterols leads to a suppression of SREBP activation. Sitosterol has been shown to directly inhibit Cyp7a1 activity in vitro and we presume this may account for the reduced enzyme activity . In preliminary studies, when knockout mice are placed on a low sitosterol diet, the activity of this enzyme, as well as the mRNA levels are increased (E Klett and S Patel, unpublished observations). Another prediction, based upon the enzyme and mRNA levels in knockout mice, is that if these mice are placed on a high cholesterol/high sitosterol diet, they may show significant accumulation of both sterols in the body. This may also be relevant to the human disorder where some, although not all affected individuals, manifest premature coronary artery disease.
Abcg8/sterolin-2 deficient mice also allow us to examine the role of this protein in biliary sterol secretion. The bile is sterol poor and upon stimulation by increasing bile salt excretion, Abcg8/sterolin-2 deficient mice cannot respond, in contrast to the wild-type mice. Thus, Abcg8/sterolin-2 is necessary for cholesterol secretion but not necessary for plant sterol secretion. It could be argued that although the biliary sterol output is comparable to that seen in wild-type mice that this is inappropriately low, since the tissue and plasma pools of sitosterol are so elevated in the knockout mice. Despite this caveat, the findings of sitosterol in significant quantities in the bile suggests that mechanisms other than via the sterolins can 'export' some of these sterols. Interestingly, although humans who are heterozygous for genetic defects in either ABCG5 or ABCG8 seem to be phenotypically normal, mice heterozygous for Abcg8/sterolin-2 deficiency show that sterol secretion is impaired, but not absent. Note that under steady-state conditions on a rodent chow diet, heterozygous animals show no significant elevations in plasma or tissue sitosterol levels, suggesting that the activity of these proteins may not be rate limiting. Furthermore, the heterozygous Abcg8+/- mice showed higher levels of biliary sitosterol relative to the wild-type mice. While the rate of total biliary sterol secretion is reduced compared to wild-type mice, since the heterozygous mice are not sitosterolemic, either the activity of these proteins is not rate limiting or other mechanisms can compensate for a 50% loss of activity. On the other hand, since feeding is intermittent, a slow, but continuous secretion during post-prandial periods could easily negate any mild temporary increase in tissue and plasma plant sterol levels in heterozygous animals. This may be amenable to testing by placing these animals on a high plant sterol diet and measuring the plasma sterol levels.
Recently, Kosters et al. reported that Abcg5/Abcg8 mRNA expression in a variety of different mouse strains correlated with biliary cholesterol secretion rates . In their studies, although diosgenin-fed mice showed a marked increase in biliary cholesterol output, mRNA levels of Abcg5 and Abcg8 were not altered. Using Western blot analyses, the protein level of Abcg5 was also not altered by diosgenin, although Abcg8 was not measured. They concluded that a parallel route for biliary cholesterol secretion might be operational, independent of the sterolins. While biliary cholesterol secretion is not completely absent in the Abcg8-deficient mice it would appear that sterolin-2 plays a major role in this process. In a genetic screen of plasma plant sterol levels, Sehayek et al. identified three loci that may be responsible for controlling plasma plant sterol levels and not one of these loci mapped to the murine STSL region . Thus, there is support for at least four loci that may be involved in regulating plasma plant sterol levels. To date the STSL locus is the only one proven to be involved in dietary sterol trafficking and the identity of the others remains to be elucidated.
We, as well as others, have proposed Abcg5/sterolin-1 and Abcg8/sterolin-2 to function as obligate heterodimers. Thus knocking out Abcg8/sterolin-2 alone is equivalent to a functional loss of both sterolins. It has been shown that Abcg8/sterolin-2 expression is required for correct apical trafficking of Abcg5/sterolin-1 to the apical surface in a polarized cell line and more recently in vivo by over-expression experiments [18, 19]. In experiments shown here, using three separately developed anti-Abcg5/sterolin-1 antibodies, we have obtained inconsistent data regarding the trafficking of Abcg5/sterolin-1 in Abcg8-/- mice. Based upon classical N-glycosylation maturation it would appear that Abcg5/sterolin-1, in the Abcg8-/- mice, does not exit the endoplasmic reticulum. However, by immunohistochemistry it appears that Abcg5/sterolin-1 is apically expressed in the Abcg8-deficient mice regardless of the anti-Abcg5/sterolin-1 antibody used. Interestingly, in a report of Abcg5/Abcg8 localization in canine gallbladder epithelial cells, these two proteins were found intracellularly under baseline conditions . But when given liver X receptor alpha (LXRα) agonist, Abcg5/sterolin-1 and Abcg8/sterolin-2 appeared to be expressed at the apical surface. In these studies the UTSW anti-Abcg5/sterolin-1 antibody was used. It is apparent with these conflicting data that the trafficking of these transporters is not as clear-cut. In a recent paper, Mangrivite et al. showed that the apical sorting of rat SPNT in polarized renal epithelial cells was independent of N-glycosylation . Therefore, at this time, we can neither exclude the possibility that Abcg5/sterolin-1 expression is functional in our Abcg8/sterolin-2 deficient mice nor can we rule it out. We have no functional assay for these proteins at present. More extensive fractionation experiments are underway to better determine the exact compartmental location of Abcg5/sterolin-1 in the Abcg8-/- mice. In this context, Plösch et al. have described an Abcg5/sterolin-1 deficient mouse that maintains the ability to secrete biliary cholesterol to the same extent as the wild-type mice and, when fed a LXR agonist, had higher Abcg8 mRNA expression and tended to secrete more biliary cholesterol than wild-type mice . It has been argued that Abcg5/sterolin-1 and Abcg8/sterolin-2 are dependent upon each other for secretion of hepatic sterols into bile . Given the data presented here and from the Abcg5-deficient mouse this does not appear to be the case. Perhaps in the previously published model a non-physiologic state has been made that generates these data. Taken together, these animal models suggest that Abcg5/sterolin-1 and Abcg8/sterolin-2 have independent function in vivo or that there are proteins other than the sterolins that can secrete biliary sterols.