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.