48 Although these results may suggest that cholesterol produced

in response to iron loading might be exported to other organs, the observation that plasma cholesterol levels showed no relationship Cytoskeletal Signaling inhibitor with either liver iron or cholesterol raises the possibility that much of the cholesterol produced by the liver under these conditions remains there. This may also explain the lack of agreement in other studies which have examined iron status and plasma levels of cholesterol. Cholesterol may also be exported directly into the canaliculus. Abcg5 is a half-transporter which dimerizes with Abcg8 to export cholesterol and plant sterols into the canaliculus,49 whereas Abcb4 is a transporter which exports cholesterol and phosphatidylcholine into the canaliculus.50 Investigation of these transporters revealed that Abcg5 mRNA correlated positively with liver iron, whereas Abcb4 mRNA correlated negatively. Superficially, this suggests up-regulation of cholesterol export into the bile, particularly given that the substrate preference for Abcb4

is phosphatidylcholine rather than cholesterol.51 selleck products However, Abcb4 knockout mice overexpressing Abcg5 and Abcg8 have only very low levels of cholesterol in the bile,52 and the presence of bile salt micelles is required to accept cholesterol.53 Thus, in the present study, despite the increase in Abcg5 transcript with increasing iron, the down-regulation of bile acid synthetic enzymes and Abcb4 mRNA under the same conditions suggests that transport of cholesterol to the bile does not increase to accommodate the increase in cholesterol production. Both iron and cholesterol metabolism are under complex regulatory control. Hence, we investigated some of the potential regulators that may explain the observed up-regulation of

cholesterol biosynthesis. Srebf2 preferentially activates many of the genes in the cholesterol biosynthesis pathway.35 In the present study, four of these genes—Hmgcr, Pmvk, Cyp51 and Sc5d—were significantly MCE up-regulated in response to increasing liver iron levels; however, the mechanism leading to this up-regulation appears to be independent of Srebf2 expression, which did not change in response to iron status. Srebf2 is regulated both transcriptionally and posttranscriptionally35 and, although we cannot rule out a posttranscriptional response of Srebf2 to iron, we believe this to be unlikely given that the majority of known targets of Srebf2 measured in the present study were not up-regulated. Similarly, expression of several genes measured in the present study—Dhcr7, Fdps, Abcg5, and Apob—is known to be regulated by CCAAT/enhancer binding protein α (C/EBPα), which is induced by iron loading.54-56 However, of these genes, only Abcg5 increased with increasing hepatic iron concentration, suggesting that C/EBPα is also unlikely to be involved in the observed up-regulation of cholesterol synthesis.