LXRs function throughout the body to control cholesterol transport, catabolism, and excretion (11
). This report describes what we believe to be the first conditional LXR-knockout mouse model constructed by selective elimination of the LXRα subtype in hepatocytes (LivKO mice). When challenged with a 2% cholesterol diet, LivKO mice accumulated increased amounts of cholesterol in liver resulting from failure to induce hepatic cholesterol excretion and catabolism, highlighting the importance of liver LXRα activity to whole body cholesterol homeostasis. Similarly, the ability of synthetic LXR agonists to stimulate biliary cholesterol excretion, inhibit fractional cholesterol absorption, and increase the output of neutral sterols in the feces was largely compromised in LivKO mice. Several recent studies have described a trans
-intestinal pathway for cholesterol excretion that bypasses biliary excretion but nevertheless can be stimulated by LXR activation (38
). Our studies suggest that such a biliary-independent pathway makes only a minor contribution to LXR agonist–dependent cholesterol excretion.
Early studies with synthetic LXR agonists described increases in plasma triglycerides and plasma HDL cholesterol as two pharmacological responses to LXR activation (19
). Analysis of LivKO mice indicates that these responses originate from unique sites. The LXR agonist–dependent increases in triglycerides were of hepatic origin and resulted from regulation of the genes encoding SREBP-1c and other enzymes involved in fatty acid and triglyceride synthesis. In contrast, hepatic deletion of LXRα had little effect on the ability of LXR agonists to elevate HDL cholesterol. Both the liver and intestine have been shown to contribute to the production of HDL (45
), and while the LXR agonist–dependent induction of Abca1
, encoding a protein required for HDL biogenesis, was impaired in liver, induction of Abca1
in the intestine was unchanged. The tissue-specific expression of Abca1
observed in LivKO mice suggests that LXR activation in the intestine is sufficient to regulate HDL cholesterol levels. Consistent with our conclusion that intestinal LXR activity is primarily responsible for elevating HDL cholesterol, previous studies indicate that expression of ABCA1 in the intestine is required for LXR agonist–dependent cholesterol increases (47
) and that transgenic overexpression of a constitutively active LXRα (VP16-LXRα) in the intestine increases HDL (48
). HDL cholesterol levels inversely correlate with cardiovascular disease risk, and the ability of LXR agonists to increase HDL cholesterol initially stimulated great interest in the therapeutic potential of such compounds (11
). The concurrent increase in lipogenesis, however, has dampened the enthusiasm for LXR agonists and slowed the progression of therapeutic molecules into the clinic. Analysis of LivKO mice demonstrates that the lipogenic and HDL pathways are tissue specific and suggests that LXR ligands that specifically target the intestine, for instance by limiting systemic absorption or by rapid first-pass clearance, could have therapeutic value.
In mouse models of cardiovascular disease, treatment with LXR agonists decreases atherosclerosis. However, in these hyperlipidemic models LXR agonists have little or no effect on HDL cholesterol levels, and this has led to the conclusion that the anti-atherogenic activity originates from increased macrophage cholesterol efflux and/or limiting inflammation in immune cells in atherosclerotic plaque (15
). Indeed, selective deletion of LXRα in hematopoietic cells increased atherosclerosis in the Ldlr–/–
background, although the increase was not as great as that measured in Ldlr–/–Lxra–/–
global knockout mice (15
). We now demonstrate that atherosclerosis was substantially increased when LXRα was selectively eliminated in hepatocytes, identifying the liver as a critical site of LXRα-dependent anti-atherogenic activity. Our studies suggest that hepatic LXRα modulates lipoprotein particle number, size, and function in a manner that influences atherogenicity. In particular, the ability of HDL to accept cholesterol from macrophages was impaired in Ldlr–/–
/LivKO mice. In addition we note that the decrease in cholesterol acceptor ability observed with HDL in Ldlr–/–
/LivKO mice correlates with the loss of the large HDL fraction observed in normal lipidemic LivKO mice. These observations suggest that pharmacological strategies utilizing small molecules that inhibit hepatic LXRα activity to reduce lipogenesis may actually increase cardiovascular disease and should be explored with caution. Future studies that examine the effect of hepatic LXR activity on lipoprotein function in the presence of the cholesteryl ester transfer protein (CETP), a lipoprotein particle–remodeling enzyme expressed in humans but not mice (56
), will be useful in this regard.
Despite the increased atherosclerosis observed in Ldlr–/–
/LivKO mice, treatment with T0901317 was still an effective preventive therapy, indicating that extrahepatic LXR activity can also be anti-atherogenic. Our in vivo RCT analysis further suggests that the ability of LXR agonists to stimulate the RCT pathway is substantially compromised in the absence of hepatic LXRα and is thus not necessary for the athero-preventive activity of LXR agonists. The efficacy of agonist treatment in LivKO mice therefore raises questions regarding the potential mechanisms and sites of action for the pharmacological activity of LXR agonists. In contrast to our findings in liver, using bone marrow transplantations we previously showed that LXR activity in hematopoietic cells is necessary for the anti-atherogenic activity of T0901317 (17
). A number of additional functions for LXRs in immune cells including the control of inflammation (2
), endoplasmic reticulum stress (57
), macrophage egress (58
), and monocyte proliferation (59
) could underlie the anti-atherogenic activity of LXR ligands. Finally, recent studies indicate that intestine-specific activation of LXRs using pharmacological or transgenic approaches can increase RCT and may beneficially impact the treatment of atherosclerosis (48
). The failure of LXR agonist treatment to increase the appearance of macrophage-derived cholesterol in the plasma of Ldlr–/–
/LivKO mice during the in vivo RCT assay further raises the possibility that impaired LXR activity in the liver can negatively affect macrophage cholesterol efflux in the periphery. The appearance of macrophage-derived 3
H-cholesterol in the plasma during the in vivo RCT assay, however, may not simply reflect the rate of the macrophage cholesterol efflux. The reentry of 3
H-cholesterol into the plasma compartment after uptake by the liver and/or intestine may also contribute to this measurement. Therefore, we cannot rule out the possibility that LXR agonists do in fact promote macrophage cholesterol efflux in Ldlr–/–
/LivKO mice and that this activity is anti-atherogenic even when hepatic cholesterol excretion to the bile is inhibited. In summary, our characterization of LivKO mice demonstrates that while endogenous hepatic LXRα activity is essential for maintaining normal lipid and sterol homeostasis, pharmacologic strategies that bypass LXR activation in liver may still be of therapeutic benefit.