Side-chain oxidized oxysterols as metabolic regulators in vivo

Sammanfattning: Oxysterols are oxygenated derivatives of cholesterol characterized by a very short half- life and their ability to pass lipophilic membranes easily, and they are considered as important intermediates in the excretion pathways of cholesterol and its conversion to bile acids. Evidence has been presented that the production and flux of oxysterols in the brain may also be of some importance for cognitive functions. The two major oxysterols in the circulation of human and mouse are 24S-hydroxycholesterol (24S- OH) and 27-hydroxycholesterol (27-OH), which are formed by the cytochrome P450 enzymes cholesterol 24-hydroxylase (CYP46A1) and sterol 27-hydroxylase (CYP27A1), respectively. The two oxysterols 27-OH and 24S-OH are both strong inhibitors of cholesterol synthesis and activators of LXR in vitro. However, their role as physiological regulators under in vivo conditions is controversial. The overall aim of this thesis was to investigate the regulatory role of side-chain oxidized oxysterols as metabolic regulators in vivo. In particular, we have studied the role of 24S-OH and 27-OH as regulators of cholesterol synthesis and activators of LXR. We used mouse models with increased levels of 27-OH like human CYP27A1 overexpressor mice (CYP27A1tg) and Cyp7b1 knockout mice (Cyp7b1-/-), as well as a mouse model with no detectable levels of 27-OH in the circulation, Cyp27a1 knockout mice (Cyp 27a1-/-). The latter mice were treated with cholic acid to compensate for the reduced formation of bile acids. Using Cyp27a1-/- mice, we have also studied the possibility that 27-OH mediates the negative effects of dietary cholesterol on memory function in mice. In Paper I, we studied a possible regulatory role of 27-OH and 24S-OH in the brain, using CYP27A1tg mice and Cyp27a1-/- mice. The levels of 27-OH were increased by approximately 12-fold in the brain of CYP27A1tg mice while levels of 24S-OH were decreased by about 25%, most probably due to increased metabolism by the CYP27A1 enzyme. Evidence was presented that cholesterol synthesis was increased in the brain of the two mouse models. There was no upregulation of the LXR-target genes in the brain of either of the two models. The increased synthesis in the brain of the Cyp27a1-/- mice is probably the consequence of the absence of an inhibitory effect of the flux of 27-OH into the brain. The increased cholesterol synthesis in the brain of the CYP27A1tg mice is probably due to the reduced levels of 24S-OH, leading to reduced inhibition of its synthesis. The results of this study are consistent with the possibility that both 24S-OH and 27-OH have a suppressive effect on cholesterol synthesis in the brain. We also conclude that 27-OH is not a general activator of LXR in this tissue. In Paper II, we examined the role of 27-OH in the liver using the three mouse models described above. Only very modest effects on cholesterol synthesis and LXR target genes were observed in the three mouse models. The overall results do not support the contention either that 27-OH is an important regulator of cholesterol homeostasis, or that 27-OH is an activator of LXR-regulated genes under basal conditions in the liver. It has been reported that treatment of mice with dietary cholesterol leads to upregulation of some LXR target genes in the liver. In a study by Chen et al, such upregulation of three different LXR target genes was not seen when treating mice lacking 24S-OH, 25-OH and 27-OH with dietary cholesterol (Chen et al., 2007). It was concluded that a 24-, 25- or 27-hydroxylation step mediated the effect of dietary cholesterol. We show here that similar effects were obtained when treating Cyp27a1-/- mice with dietary cholesterol, suggesting that the effects are mediated by 27-OH. Most of these effects could however not be observed at either the protein level, or at the level of enzyme activity. The results suggest that 27-OH is a mediator of the cholesterol-induced effects on some LXR target genes when the mice are challenged with a high load of dietary cholesterol. The physiological importance of this effect is difficult to evaluate. In Paper III we compared the two mouse models CYP27A1tg and Cyp7b1-/- mice, both of which have high levels of 27-OH in the circulation and the brain. In contrast to CYP27A1tg mice, the levels of 24S-OH in the brain were not decreased in the latter model, and cholesterol synthesis was not affected. This supports the proposal that 24S- OH is of regulatory importance for cholesterol synthesis in the brain. It has been reported that cholesterol synthesis is reduced in the kidney of Cyp7b1-/- mice. Cholesterol synthesis in the kidney of CYP27A1tg mice was not affected, however. We conclude that factors other than high levels of 27-OH are of importance for the reduced cholesterol synthesis in the kidney of the Cyp7b1-/- mice. In Paper IV we tested the possibility that the negative effect of dietary cholesterol on cognition in mice is mediated by 27-OH. The negative effect of dietary cholesterol on spatial memory observed in wild-type mice was not observed in Cyp27a1-/- mice. The latter mice were also treated with cholic acid to compensate for the reduced synthesis of bile acids. Treatment with dietary cholesterol was shown to lead to reduced levels of the “memory protein” Arc (Activity-Regulated Cytoskeleton-associated protein) in the hippocampus of the wildtype mice. This effect was not seen in the hippocampus of Cyp27a1-/- mice. The results are consistent with the possibility that 27-OH is a mediator of the negative effects of dietary cholesterol on cognition. In conclusion, our results suggest that both 24S-OH and 27-OH may be of some regulatory importance for cholesterol synthesis in the brain but not in the liver. Under normal basal conditions 27-OH does not seem to be a general activator of LXR, either in the brain or in the liver. We discuss the possibility that the different effects on cholesterol synthesis in the two organs may be related to the fact that almost all oxysterols in the brain are in the free form, whereas in the liver, most of them are esterified. In addition, we demonstrate that the negative effects of dietary cholesterol on cognition is mediated by 27-hydroxylation.