The liver X receptor: Control of cellular lipid homeostasis and beyond

Progress in Lipid Research 49 (2010) 343–352

Contents lists available at ScienceDirect

Progress in Lipid Research journal homepage: www.elsevier.com/locate/plipres

Review

The liver X receptor: Control of cellular lipid homeostasis and beyond Implications for drug design Maaike H. Oosterveer a,*, Aldo Grefhorst a, Albert K. Groen a,b, Folkert Kuipers a,b a

Department of Pediatrics, Center for Liver Digestive and Metabolic Diseases, University Medical Center Groningen, University of Groningen, P.O. Box 30.001, 9700 RB, Groningen, The Netherlands Department of Laboratory Medicine, Center for Liver Digestive and Metabolic Diseases, University Medical Center Groningen, University of Groningen, P.O. Box 30.001, 9700 RB, Groningen, The Netherlands b

a r t i c l e

i n f o

Article history: Received 12 January 2010 Received in revised form 22 February 2010 Accepted 19 March 2010

Keywords: LXR Atherosclerosis Lipogenesis Reverse cholesterol transport Drug design

a b s t r a c t Liver X receptor (LXR) a and b are nuclear receptors that control cellular metabolism. LXRs modulate the expression of genes involved in cholesterol and lipid metabolism in response to changes in cellular cholesterol status. Because of their involvement in cholesterol homeostasis, LXRs have emerged as promising drug targets for anti-atherosclerotic therapies. In rodents, synthetic LXR agonists promote cellular cholesterol efflux, transport and excretion. As a result, the progression of atherosclerosis is halted. However, pharmacological LXR activation also induces hepatic steatosis and promotes the secretion of atherogenic triacylglycerol-rich VLDL particles by the liver, complicating the clinical application of LXR agonists. The more recently emerged roles of LXRs in fat tissue, pituitary and brain may have implications for treatment of obesity and Alzheimer disease. In addition to the improvements in atherosclerosis, LXR activation exerts beneficial effects on glucose control in mouse models of type 2 diabetes. Future therapeutic strategies aiming to exert beneficial effects on cholesterol and glucose homeostasis, while circumventing the undesired effects on hepatic lipid metabolism, should target specific LXR-mediated processes. Therefore, tissue and/or isotype-specific effects of LXR action need to be established. The consequences of combinatorial drug approaches and the identification of the co-regulatory networks involved in the LXR-mediated control of particular genes may contribute to development of novel LXR agonists. Finally, pathway analyses of LXR actions provide tools to evaluate and optimize the effectiveness of novel therapeutic strategies to prevent and/or treat metabolic diseases. Ó 2010 Elsevier Ltd. All rights reserved.

Contents 1. 2.

3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . LXRa and LXRb are nuclear receptors that act as cellular cholesterol sensors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Determinants of nuclear receptor activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Physiological regulation of LXRs actions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Contributions of transgenic models and pharmacological ligands to LXR research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modulation of lipid homeostasis by LXR agonists: a potential anti-atherosclerotic therapy? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Role of LXRs in control of plasma lipid concentrations and bile acid metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Hepatic steatosis upon pharmacological LXR activation: a drawback for the clinical application of LXR agonists?. . . . . . . . . . . . . . . . . .

344 344 344 344 344 345 345 346

Abbreviations: ABC, ATP binding cassette; ACAT, acyl-CoA:cholesterol O-acyltransferase; ACTH, adrenocorticotropic hormone; Angptl3, angiopoietin-like protein 3; Apo, apolipoprotein; CA, co-activator; CE, cholesteryl-ester; CETP, cholesteryl-ester transfer protein; ChREBP, carbohydrate response element binding protein; CR, co-repressor; CYP7A1, cholesterol 7-alpha-hydroxylase; DHCR24, 3-beta-hydroxysteroid-delta 24 reductase; FAS, fatty acid synthase; FoxO1, forkhead box o1; FXR, farnesoid X receptor; G6Pase, glucose-6-phosphatase; HDL, high-density lipoprotein; HPA, hypothalamus–pituitary–adrenal; IRS, insulin receptor substrate; LPL, lipoprotein lipase; LXR, liver X receptor; NEFA, non-esterified fatty acid; Npc1L1, niemann-pick C1 Like 1; PGC, peroxisome proliferator activated receptor gamma co-activator; PDK, pyruvate dehydrogenase kinase; PEPCK, phosphoenolpyruvate carboxykinase; PLTP, phospholipid transfer protein; PPAR, peroxisome proliferator activated receptor; PXR, pregnane X receptor; RCT, reverse cholesterol transport; RE, response element; RXR, retinoid X receptor; SCD, stearoyl-CoA desaturase; SREBP, sterol regulatory element binding protein; TICE, trans-intestinal cholesterol excretion; TAG, triacylglycerol; UCP, uncoupling protein; VLDL, very-low density lipoprotein. * Corresponding author. E-mail addresses: [email protected] (M.H. Oosterveer), [email protected] (F. Kuipers). 0163-7827/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.plipres.2010.03.002

344

4.

5.

6. 7.

8.

M.H. Oosterveer et al. / Progress in Lipid Research 49 (2010) 343–352

3.3. CETP as a modulator of LXRs actions on lipoprotein metabolism? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modulation of glucose homeostasis: is LXR a target for diabetes treatment? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. LXR and hepatic glucose metabolism: reduced gluconeogenesis upon LXR activation?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. The role of LXR in insulin action and lipid metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other physiological modes of LXR action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. LXR as a regulator of adipocyte energy homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. LXRs in control of steroidogenesis in the pituitary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. LXRs regulate cholesterol metabolism in the brain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pitfalls and challenges in LXR research. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Implications and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Modulation of LXRs co-regulatory complex. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Combinatorial drug therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The liver X receptor (LXR), was discovered in 1995 as a new member of the nuclear receptor superfamily [1], and found to be activated by oxygenated cholesterol derivatives [2]. Since then, LXR research has been booming and prominent roles of this nuclear receptor in control of cholesterol, bile acid and lipoprotein metabolism, but also in fatty acid, triacylglycerol and glucose homeostasis have emerged. In addition, LXRs appear to play an important role in regulation of inflammation and intestinal lipid transport. Most of these aspects of LXR action have been topics of recent outstanding reviews [3–5]. The aim of the present report is to provide a picture of LXR as an integrator of different physiological processes since, despite the plethora of its metabolic actions, the LXR-system should be considered as an interesting drug target. 2. LXRa and LXRb are nuclear receptors that act as cellular cholesterol sensors 2.1. Determinants of nuclear receptor activity LXR is a member of the nuclear receptor superfamily which comprises various sub-families that share the ability to promote or inhibit the transcription of target genes by binding to specific sites in the promoter regions of these genes, the so-called response elements. Most nuclear receptors, including LXR, act as ligand-activated transcription factors. Initiation or blockade of target gene transcription is determined by the presence of coactivator or co-repressor complexes [6]. The affinity for and the composition of these regulatory complexes depend on the structural conformation of the nuclear receptor, which is altered upon ligand binding but also by post-translational modifications [7]. Thus, transcriptional control by a nuclear receptor is a complex process and regulated at multiple levels as is schematically summarized in Fig. 1.

346 347 347 347 348 348 348 348 348 349 349 349 349 349 349

ing oxysterols are 22(R)-hydroxycholesterol and 20(S)-hydroxycholesterol, which are intermediates in steroid hormone synthesis. Other ligands are 24(S)-hydroxycholesterol, that is produced in the brain and represents the most abundant circulating oxysterol, and 24(S),25-epoxycholesterol which is mainly found in the liver [9]. All but one of these oxysterols species [10] have been reported to activate both LXRa and LXRb [2,9,11–13]. Interestingly, it was shown that glucose and glucose-6-phosphate are able to bind and activate LXR in vitro [14]. However, the physiological relevance of this observation has been debated [15–17]. LXR-mediated gene regulation requires its heterodimerization with another nuclear receptor, the retinoid X receptor (RXR) which is ligand-activated by 9-cis retinoic acid. The consensus LXR response element (LXRE) consists of two hexameric nucleotide direct repeats separated by four nucleotides (DR4: consensus sequence: 50 -AGGTCAnnnnCGGTCA-30 ) [1]. LXRs interact with several coregulators including PGC-1b, RIP140, GPS2 and ACS-2, which have been linked to specific metabolic processes [18–21]. In addition, LXR activity is determined by its phosphorylation, acetylation and/or SUMOylation status [20,22–28]. A very recent study shows that LXRs are also subject to O-linked N-acetylglucosaminylation [29], likely explaining their glucose-sensing actions [14,30]. Finally, part of the LXR promoter is hypermethylated upon prenatal protein restriction [31], representing an epigenetic mode of action to modify LXR activity. Polyunsaturated fatty acids repress LXR activity in vitro [32,33] while insulin stabilizes LXR mRNA expression [34]. The latter results and those reported by Mitro et al. [14] point towards an important regulatory role of LXR in the integration of fatty acid and glucose metabolism. The identification of cholesterol metabolites as endogenous ligands already hints toward the physiological processes that are regulated by LXRs. Upon their activation, LXRs induce the transcription of multiple genes involved in cholesterol efflux, conversion and transport [35]. However, LXRs also modulate the expression of genes involved in glucose and fatty acid metabolism while transrepressing those involved in inflammatory processes [3].

2.2. Physiological regulation of LXRs actions Two LXR isotypes have been identified: LXRa (NR1H3) that is mainly expressed in the liver and to a lesser extent in intestine and adipose tissue, and LXRb (NR1H2) that is ubiquitously expressed [1,8]. The physiological LXR ligands are oxysterols, the oxygenated metabolites of cholesterol. Because cellular oxysterol concentrations reflect cellular cholesterol content, LXRs act as cholesterol sensors that induce adaptive physiological changes in response to cellular cholesterol overload. Among the activat-

2.3. Contributions of transgenic models and pharmacological ligands to LXR research Because of their regulatory roles in important metabolic pathways, LXRs have been identified as potentially interesting drug targets to adjust disturbances in lipid and glucose metabolism that predispose to development of metabolic diseases such as atherosclerosis and type 2 diabetes. Although their activity is regulated at multiple levels, so far most therapeutical strategies aim to mod-

345

M.H. Oosterveer et al. / Progress in Lipid Research 49 (2010) 343–352

1. Ligand-binding LXR: oxysterols RXR: 9-cis retinoid acid

RXR 2. Nuclear translocation

LXR

3. Receptor dime rization

4. Post-translational modifications 5. Co-activ ator rec ruitment histone acetylation

CR

histone methylation

CA

SUMO-ylation phosphorylation

LXRE RXRE

LXRE RXRE transcription

Fig. 1. Regulatory steps that determine LXR’s transcriptional activity. (1) LXRs are ligand-activated by oxysterols. (2) LXR’s transcriptional regulation requires its translocation to the nucleus. and its (3) heterodimerization with 9-cis retinoid acid-activated RXR. LXR and RXR bind to specific response elements (RE) on the DNA. Ligand binding and (4) post-translational modifications alter the structural conformation of the LXR/RXR complex, thereby modifying the affinity for certain (5) co-repressor (CR) or co-activator (CA) proteins that determine whether a target gene is induced or suppressed.

ulate LXRs actions via ligand-activation of the receptors. The majority of LXR research has been performed with the synthetic ligands T0901317, GW3965 or WAY252623. Because of the recruitment of different co-regulatory proteins, these agonists may exert tissue-specific effects [36]. The use of pharmacological LXR agonists in vitro and in vivo and the generation of isotype-specific and LXRab double knockout mice has significantly contributed to the unraveling of LXRs metabolic actions. The experimental data obtained indeed provide a rationale to target LXRs for prevention and/or treatment of metabolic diseases. The putative and therapeutic relevant metabolic targets of LXR agonists are discussed below, with emphasis on the roles of LXR in atherosclerosis and diabetes.

3. Modulation of lipid homeostasis by LXR agonists: a potential anti-atherosclerotic therapy? 3.1. Role of LXRs in control of plasma lipid concentrations and bile acid metabolism The most commonly used synthetic LXR agonists have all been shown to exert beneficial actions in atherosclerosis-prone mouse models [37–44]. The reduction in atherosclerotic plaques upon pharmacological LXR activation results from a reduction in intestinal cholesterol absorption [45–48] and a concomitant induction of multiple steps in the reverse cholesterol transport (RCT) pathway [49–51].

346

M.H. Oosterveer et al. / Progress in Lipid Research 49 (2010) 343–352

By inducing the expression of the genes encoding ATP binding cassette A1 and G1 (ABCA1 and ABCG1), LXR promotes cholesterol efflux to apolipoprotein A-I (ApoA-I) and high density lipoprotein (HDL) [52,53] from, among others, (lipid-loaden) macrophages [54,55]. Cellular cholesterol efflux is considered to be the first step in RCT. LXR not only regulates ABCA1 at the transcriptional level [56] but also by post-translational means since inactive LXRb binds to ABCA1 [57]. Both hepatic and intestinal ABCA1 are required for HDL formation [58,59] and intestinal ABCA1 contributes to the increase in plasma HDL upon pharmacological LXR activation [60]. Interestingly, GW3965 fails to increase plasma HDL in mice deficient for Niemann-Pick C1 Like 1 (Npc1L1), a transporter crucial for intestinal cholesterol absorption [61]. This suggests that intestinal cholesterol absorption contributes to the increase in HDL-cholesterol upon pharmacological LXR activation. LXR agonists not only induce HDL-cholesterol levels and size [62], but also alter their metabolism via induction of the genes encoding phospholipid transfer protein (PLTP), lipoprotein lipase (LPL) and cholesteryl-ester transfer protein (CETP) [63–65]. PLTP facilitates phospholipid transfer to HDL and may enable HDL remodeling [66,67]. LPL mediates triacylglycerol (TAG) hydrolysis and is essential for peripheral fatty acid uptake from TAG-rich lipoproteins. LPL has also been implicated in HDL uptake [68,69]. CETP facilitates cholesteryl-ester (CE) and TAG exchange between HDL and apoB-containing lipoproteins [70], hence modulating HDL-levels and size [71]. HDL mediates cholesterol transport from peripheral tissues to the liver. Hepatic LXR induces cholesterol conversion into bile acids, via induction of the gene encoding cholesterol 7-alphahydroxylase (CYP7A1) in murine [9] but not human liver cells [72]. LXR also enhances biliary cholesterol secretion [48,73] by inducing the heterodimer Abcg5/Abcg8, which mediates cholesterol secretion from the liver into the bile [46]. Interestingly, the increase in biliary and fecal cholesterol excretion is unaltered upon pharmacological LXR activation in Abca1-deficient mice lacking HDL [73]. This suggests that other pathways may compensate for the loss in Abca1-induced cholesterol efflux. Steffensen and Gustafsson discussed the supposedly specific roles of the two LXR isotypes in lipoprotein metabolism by comparing the phenotypes of LXRa, LXRb and LXRab double knockout mice [74]. The expression of genes involved in hepatic cholesterol metabolism was found to be reduced in LXRa-deficient mice. LXRb-deficient mice, on the other hand, failed to show these alterations, consistent with a more prominent role of LXRa compared to LXRb in the regulation of lipoprotein metabolism. Recently, trans-intestinal cholesterol excretion (TICE), i.e., the direct secretion of circulating cholesterol into the intestinal lumen [75,76], has emerged as the major contributor to LXR-induced fecal cholesterol excretion [50]. TICE is reduced in Abcg5-deficient mice [75]. Furthermore, the induction of RCT upon pharmacological LXR activation is abolished in Abg5/8-deficient mice. Together, these data suggest that intestinal expression of the cholesterol transporter-dimer Abcg5/g8 contributes to TICE [51]. TICE contributes significantly to fecal cholesterol excretion [50] and is increased upon knockdown of acyl-CoA:cholesterol O-acyltransferase 2 (ACAT2) [77], which mediates cholesterol esterification. The importance of TICE in cholesterol excretion is supported by the observation that the LXR-mediated increase in fecal sterol excretion is maintained when biliary cholesterol secretion is impaired [48]. However, more research is needed to reveal the exact mechanisms underlying TICE. Besides promoting RCT, a very recent report indicates that LXR activation may reduce cellular cholesterol uptake by enhancing the ubiquitination of the LDL-receptor [78], thereby accelerating LDL-receptor degradation. Finally, LXR agonists slow down atherosclerosis progression by suppressing inflammatory processes within the arterial wall [3]. The LXR-

mediated increase in HDL-levels may also contribute to antiinflammatory actions [79], and depend on enhanced cholesterol turnover via 3 beta-hydroxysteroid-delta 24 reductase (DHCR24) [80]. 3.2. Hepatic steatosis upon pharmacological LXR activation: a drawback for the clinical application of LXR agonists? Although LXRs have emerged as a potential powerful anti-atherosclerotic drug target, their involvement in hepatic fatty acid and TAG metabolism provokes serious concerns for their clinical application. LXR agonists induce fatty liver and promote the secretion of large, TAG-rich VLDL particles in mice [62,81]. The enhanced secretion of VLDL-TAG is paralleled by an increase in Angiopoietin-like protein 3 (Angptl3) expression and secretion [82]. Angptl3 inhibits LPL activity and increases plasma TAG and HDL cholesterol concentrations [83]. Although no quantitative data are available on the rate of fatty acid synthesis upon pharmacological LXR activation, the development of fatty liver has been attributed to an induction of fatty acid synthesis and their subsequent incorporation into TAGs. It has however also been reported that the induction of the fatty acid transporter CD36 in response to pharmacological LXR activation contributes to hepatic lipid accumulation, presumably by increasing hepatic fatty acid uptake [84]. The induction of lipogenic genes by LXR agonists is largely mediated by the LXR target gene encoding for sterol regulatory element binding protein 1c (SREBP-1c) [85], but partly also directly by LXR itself [86]. For instance, LXR agonist treatment has been shown to induce the genes encoding fatty acid synthase (FAS) and stearoylCoA desaturase 1 (SCD1) in Srebp-1c-deficient mice [86]. The direct regulation of lipogenic genes by LXR may represent an adaptive response to cellular cholesterol overload, because it enables the formation of relatively harmless CEs [81]. LXR increases Srebp-1c expression and its nuclear translocation [87] while Srebp-1c translocation is also promoted by insulin [88]. Besides Srebp-1c, LXR has been reported to increase the expression of the lipogenic transcription factor carbohydrate response element binding protein (Chrebp) [89], while Chrebp activity is enhanced by an increased glucose availability [16]. Altogether, by controlling both Srebp-1c and Chrebp transcription, LXR contributes to the induction of fatty acid synthesis in response to glucose and insulin [16]. LXR agonist treatment may therefore have more drastic consequences for hepatic lipid content in type 2 diabetic patients, that exhibit both hyperglycaemia and hyperinsulinemia. However, quantitative analysis of the lipogenic flux in humans is required to establish the clinical relevance of the increased hepatic lipogenesis observed in rodents receiving LXR agonists. Interference with the lipogenic ability at the level of Srebp-1c and Scd1 protects against the fatty liver induced by LXR agonists [90]. Furthermore, LXRa-deficient mice do not develop hepatic steatosis when treated with an agonist that activates both isotypes [91]. It is currently unknown whether the anti-atherosclerotic properties of LXR are also maintained under these conditions. On the other hand, the lipogenic induction upon pharmacological LXR activation may protect against lipotoxicity in b-cells and endothelium, because the increased lipogenic flux may facilitate the storage of free fatty acids as TAGs [92,93]. 3.3. CETP as a modulator of LXRs actions on lipoprotein metabolism? Basciano et al. showed increased apoB synthesis and -secretion by hepatocytes of hamsters treated with an LXR agonist while TAG secretion was unaffected [94]. These data suggest that the secreted very low density lipoprotein (VLDL) particles were smaller under these conditions. This was associated with elevated plasma VLDL-TAG concentrations in LXR-agonist treated hamsters. VLDL

347

M.H. Oosterveer et al. / Progress in Lipid Research 49 (2010) 343–352

cholesterol absorption ↓

fatty acid synthesis ↑ triglyceride storage ↑

biliary cholesterol secretion ↑

TG TG TG

? HDL ApoA1

cholesterol transport FECAL CHOLESTEROL EXCRETION ↑

CETP ↑ VLDL

TICE ↑

cholesterol efflux ↑ macrophage

Fig. 2. Consequences of pharmacological LXR activation for lipid homeostasis. LXR agonists reduce intestinal cholesterol absorption, and promote reverse cholesterol transport by inducing cellular cholesterol efflux, cholesterol transport and cholesterol secretion via the bile. As a result, fecal cholesterol excretion is enhanced. LXR agonists furthermore increase hepatic lipid synthesis and enhance CETP activity.

cholesterol content was also increased, probably due to the increased synthesis and secretion of CEs. These results are in sharp contrast to those obtained in LXR-agonist treated mice, that have been shown to secrete large, TAG-rich VLDL particles [62,81,95]. Because hamsters, in contrast to mice, express CETP, the differences in response to pharmacological LXR activation are most likely related to CETP. Recently, Quinet et al. [43] investigated the differential effects of LXR activation in Cynomolgus monkeys. Treatment of these primates, that do express CETP, with WAY252623 resulted in decreased plasma cholesterol concentrations, which was mainly due to a reduction in plasma LDL cholesterol. Furthermore, CETP overexpression in mice attenuates the increase in biliary cholesterol secretion in response to LXR agonist treatment [96]. Although these data suggest that CETP does play an important role in the LXRmedicated effects on plasma lipids, the anti-atherosclerotic actions of LXR agonists are presumably maintained in CETP-expressing species, since a recent report showed an induction of RCT upon pharmacological LXR activation in hamsters [97]. The aforementioned physiological adaptations in lipid homeostasis that occur upon pharmacological LXR activation are summarized in Fig. 2. 4. Modulation of glucose homeostasis: is LXR a target for diabetes treatment? 4.1. LXR and hepatic glucose metabolism: reduced gluconeogenesis upon LXR activation? LXR agonists have been shown to improve glycemic control in diabetic rodent models [98–101]. On the other hand, LXR agonists do not affect blood glucose concentrations in normoglycemic animals [98,100]. Hyperinsulinemic euglycemic clamp studies, considered as the ‘gold standard’ to determine insulin sensitivity in vivo [102], correspondingly showed that insulin sensitivity is not affected in LXRa-deficient and LXRb-deficient mice or lean, normoglycemic mice treated with an LXR agonist [17,100]. On the other hand, LXR agonists clearly improved insulin sensitivity in diabetic rodent models, i.e., ob/ob mice and high-fat fed rats [100,101]. These data indicate that insulin resistance of glucose metabolism is partially corrected upon LXR activation. More specifically, LXR agonist treat-

ment improved peripheral but not hepatic insulin resistance in ob/ob mice [100]. Furthermore, it did not affect hepatic insulin resistance in high-fat fed rats [101]. Thus, pharmacological LXR activation results in tissue-specific improvements in insulin sensitivity, which appear to depend on the underlying pathophysiological mechanism of insulin resistance. LXR activity appears to affect the expression of genes encoding the gluconeogenic enzymes phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase) and peroxisome proliferator activated receptor gamma co-activator 1a (PGC1a) [17,98,99,101]. However, these changes do not affect the gluconeogenic flux in vivo, since neither pharmacological LXR activation nor LXR deficiency affected endogenous glucose production in both non-diabetic and ob/ob mice [17,100]. 4.2. The role of LXR in insulin action and lipid metabolism LXR agonists promote insulin secretion by pancreatic b-cells [104] and human islets [105]. However, a major effect on circulating insulin concentrations in vivo has not been reported. Some studies show slightly increased plasma insulin concentrations upon LXR activation [100]. Phosphorylated insulin receptor substrate 1 (IRS1) protein expression has been reported to be reduced in livers from LXR-agonist treated hamsters, while the expression of phosphorylated IRS2 protein was increased [94]. Previously, it was shown that the hepatic expression of the protein forkhead box o1 (FoxO1), a downstream target of the insulin signaling cascade, is reduced upon pharmacological LXR activation [95]. While in the latter study FoxO1 expression was determined under hyperinsulinemic euglycemic clamp conditions, the hamster experiments by Basciano et al. did not interrogate the direct effects of insulin on IRS1mediated insulin signaling. Therefore, it is not clear whether the reduced expression of IRS1 upon LXR agonist treatment resulted in insulin resistance of hepatic lipid and/or glucose metabolism in the liver of LXR-agonist treated hamsters. The reduced ability of insulin to suppress VLDL-TAG secretion upon pharmacological LXR activation [95] may represent a feature of impaired IRS1mediated regulation of hepatic lipid metabolism. On the other hand, this phenotype may also be secondary to an increase in hepatic lipid synthesis upon pharmacological LXR activation.

348

M.H. Oosterveer et al. / Progress in Lipid Research 49 (2010) 343–352

Although insulin has been suggested to activate LXR [34], hyperinsulinemia did not result in an additional increase in lipogenic gene expression in mice treated with an LXR agonist [95]. It is also debatable whether insulin actually increases SREBP-1c via LXR [34]. Thus, whether or not insulin interferes with the induction of lipogenic genes upon pharmacological LXR activation remains to be established. Taken together, despite the great body of evidence linking pharmacologial LXR activation to hepatic lipid synthesis and the development of fatty liver, more research is needed to reveal the exact underlying mechanisms. 5. Other physiological modes of LXR action 5.1. LXR as a regulator of adipocyte energy homeostasis Besides the intensive research on LXRs actions in control of RCT and lipid metabolism, evidence directing towards a pivotal role of LXRs in adipocyte biology is accumulating. LXRb-deficient and LXRab double knockout mice have small adipocytes, which do not increase in size upon aging or high-fat feeding [103,106]. LXR agonist treatment of differentiated adipocytes enhanced glucose uptake and glycogen synthesis in vitro but also increased non-esterified fatty acid (NEFA) release by these cells, suggesting increased lipolysis [107]. This is in line with in vivo data from the same report showing a time-dependent increase in plasma NEFA and glycerol concentrations upon pharmacological LXR activation. The elevated plasma NEFA concentrations have also been observed in a separate study upon T0901317 treatment [62]. NEFA concentrations were, however, not increased in GW3965-treated mice [100]. The increased glucose uptake by adipocytes in response to LXR activation is thought to result from an induction of both GLUT1 and GLUT4 expression [99]. In another study, it was shown that adipose tissue GLUT4 expression was induced upon GW3965 treatment in ob/ob mice but not in lean mice [100]. The LXR agonist GW3965 has been shown to promote adipocyte fatty acid oxidation in vitro in a time- and dose-dependent manner [108]. Furthermore, this agonist increased the expression and activity of pyruvate dehydrogenase kinase 4 (PDK4) under these conditions [108]. PDK4 inhibits the pyruvate dehydrogenase complex and hence reduces glucose oxidation. Both LXRa and LXRb have also been implicated in the regulation of uncoupling protein 1 (UCP1) expression in adipose tissue [103,106]. Activated LXRa suppresses UCP1 expression [109]. These data suggest that LXRs control energy dissipation, and determine the amount of energy available for fat storage [106,110]. So far, experimental evidence for a direct association between adipocyte UCP1 expression and susceptibility to diet-induced obesity is inconsistent [106,110] and requires further investigation. Altogether, these data suggest a regulatory role of LXR in adipose tissue substrate oxidation. There is however currently no evidence that LXR agonists lower energy expenditure and/or increase fat storage in vivo. Genome-wide expression profiling experiments investigating the effects of pharmacological LXR activation in LXRab double knockout mice and their wild-type littermates showed that upon LXR agonist treatment, LDL-receptor gene expression is up-regulated in adipose tissue in an LXR-dependent fashion [111], while Zelcer et al. showed reduced expression of the LDL-receptor protein upon pharmacological LXR activation in small intestine and macrophages [78]. This reduction was attributed to the induction of inducible degrader of the LDLR (IDOL), which promotes LDLR ubiquitination. 5.2. LXRs in control of steroidogenesis in the pituitary The genome-wide expression profiling upon pharmacological LXR activation also uncovered major changes in adrenal gene

expression levels [112]. One week of LXR agonist treatment in mice induced adrenocorticotropic hormone (ACTH) receptor gene expression in the adrenal gland while UCP1, UCP3 and glycolytic enzyme expression was suppressed. Furthermore, plasma corticosterone levels were elevated. However, a reduction in adrenal steroid hormone production was observed upon LXR agonist treatment of adrenal cells in culture [113]. These differential consequences for steroidogenesis are probably related to the concomitant effects of LXR on the hypothalamus–pituitary–adrenal (HPA) axis in vivo. This is supported by the up-regulation of ACTH receptor expression in the adrenal gland and the elevated ACTH plasma concentrations in mice treated with an LXR agonist [112]. More strikingly, it has been suggested that plasma HDL concentrations modulate adrenal gland steroidogenesis by inducing cholesterol transport into the adrenals, hence providing the substrate needed for steroid hormone synthesis [114]. The elevated plasma corticosterone concentrations upon pharmacological LXR activation might therefore also be secondary to the elevated plasma HDL-levels, possibly in combination with the proposed effects on ACTH. Yet, elucidation of the exact mechanism of action requires additional detailed investigation. LXR’s interference with the HPA axis may also impact on energy metabolism, since elevated plasma corticosterone concentrations have been shown to alter glucose metabolism in liver and peripheral tissues [115].

5.3. LXRs regulate cholesterol metabolism in the brain Seladin-1/DHCR24 is a key enzyme in the cholesterogenic pathway. Reduced expression of this enzyme in certain brain regions is associated with Alzheimer’s disease [116]. Interestingly, Seladin-1/ DHCR24 is a LXR target gene [117] and pharmacological LXR activation improved disease pathology, memory function and led to increased brain cholesterol turnover in mouse models of Alzheimer’s disease [118–122].

6. Pitfalls and challenges in LXR research The major pitfall of pharmacological LXR activation is undoubtedly the development of fatty liver in both rodents and primates [43,62,100]. Yet, current views on the metabolic consequences of pharmacological LXR activation may not be complete and entirely correct, since the commonly used LXR agonists may exert some aspecific effects [123,124]. For instance, the suppression of G6Pase expression upon T0901317 treatment has been shown to result from an increased activity of ROR instead of LXR [124] and the concomitant activation of the pregnane X receptor (PXR) may in part contribute to the development of fatty liver in T0901317-treated animals [123,125]. On the other hand, the HDL-raising effect of T0901317 is most likely independent of PXR activation [126]. The majority of the experimental evidence directing toward the anti-atherosclerotic properties of LXR agonist have been performed in rodents and only very few studies have been conducted in species with a human-like lipoprotein profile and/or that express CETP [41,43,94,97,127], although CETP modulates HDL metabolism and biliary cholesterol secretion upon pharmacological LXR activation [71,96]. LXR agonists are able to induce RCT in hamsters [97]. CETP has furthermore been shown to increase LDL in hamsters and Cynomolgus monkeys [127]. To date, only one report on the effects of pharmacological LXR activation in humans has been published [128]. However, in this study, potential effects on atheroclerosis have not been evaluated. Therefore, the clinical relevance of LXR agonist for treatment of cardiovascular diseases remains to be established.

M.H. Oosterveer et al. / Progress in Lipid Research 49 (2010) 343–352

349

7. Implications and future directions

8. Concluding remarks

From this overview of LXRs’ metabolic actions it is evident that LXR plays an important role in various metabolic pathways, but it is also acting in multiple tissues. Tissue and/or isotype-specific targeting of LXR has repeatedly been proposed [3,5129,130] as a potentially effective strategy to promote RCT while circumventing dyslipidemia and fatty liver. Tissue-specific effects of nuclear receptor agonists including those activating LXR have been reported [131–133]. Experiments involving transgenic mouse models revealed that LXRb is the major isotype controlling energy and glucose metabolism, while LXRa is mainly involved in the regulation of hepatic lipid metabolism [106]. It is unclear whether these differential actions are related to specific downstream effects of both isotypes or to their differential tissue expression. Ideally, pharmacological agonists should be specifically targeted to LXRb in macrophages, and promote cholesterol efflux from these cells without inducing lipogenesis in the liver. Data on the effects of selective LXRb agonists are currently not available. Treatment of LXRa-deficient mice with a non-selective LXR agonist provides an alternative: these treated animals did not develop a fatty liver. However, plasma VLDL-TAG concentrations were markedly elevated [91] in LXRa-deficient mice treated with the LXR agonist. More studies are required to assess the underlying mechanisms of these findings. Finally, if intestinal Abcg5/8 is really the major contributor to cholesterol excretion via TICE [50,51] and if intestinal LXR plays crucial regulatory role in TICE, intestine-specific LXR activation may effectively reduce plasma LDL cholesterol concentrations and hence protect against atherosclerosis.

Many of the metabolic processes controlled by LXRs are interlinked. When LXR is considered as a putative drug target one should study its actions from a whole-body perspective. Although in vitro studies as well as organ-specific transcriptomic and proteomic profiling might be helpful to identify the metabolic pathways regulated by LXRs, the actual physiological effects of pharmacological LXR activation should ideally be studied in intact organisms using a fluxomics approach [140], i.e., using experimental settings in which the actual fluxes through metabolic pathways are quantified. Furthermore, adequate insight into the effects of LXR agonists on the different metabolic pathways and their complex interactions requires a systems biology approach. In this respect, metabolic pathway analysis of the global consequences of pharmacological LXR activation may be particularly helpful [141,142].

7.1. Modulation of LXRs co-regulatory complex Another strategy to exclusively target the reverse cholesterol transport pathway is via modulation of the co-regulatory complex [19,134] since the different co-activators and co-repressors are associated with specific downstream effects of LXRs [19,20, 109,135]. The recruitment of different co-regulatory proteins has also been suggested to contribute to tissue-specific responses to various synthetic LXR agonists [36]. The LXR-mediated induction of lipogenic genes depends on the presence of its co-repressor RIP140 [19]. Recent data indicate an increased expression of another member of LXRs co-activator complex, ASC-2, in macrophages versus liver. However, the exact contribution of ASC-2 to RCT and hepatic lipogenesis needs to be established. Because the composition of co-regulator complexes depends on the structural conformation of the nuclear receptor, possibilities to interfere with post-translational processes must also be explored [22–27]. 7.2. Combinatorial drug therapy Synergistic drug combinations may counteract the undesired side effects of LXR agonists [136]. Although multi-drug approaches aiming to activate multiple nuclear receptor activities and thereby counteract hepatic fat accumulation have so far not proven to be very successful [137], there are still a number of interesting strategies that remain to be investigated. Co-treatment with an LXR and peroxisome proliferator activated receptor a (PPARa) agonists resulted in an additional increase in plasma HDL concentrations but did not reduce hepatic steatosis [137], presumably because PPARa agonists, besides promoting fat oxidation, have also been shown to induce hepatic lipogenesis [138]. The simultaneous activation of LXR and the farnesoid X receptor (FXR) may be more promising, since the latter has been shown to reduce hepatic lipogenesis [139].

Conflict of interest The authors declare no conflict of interest.

References [1] Willy PJ, Umesono K, Ong ES, Evans RM, Heyman RA, Mangelsdorf DJ. LXR, a nuclear receptor that defines a distinct retinoid response pathway. Genes Dev 1995;9:1033–45. [2] Janowski BA, Willy PJ, Devi TR, Falck JR, Mangelsdorf DJ. An oxysterol signalling pathway mediated by the nuclear receptor LXR alpha. Nature 1996;383:728–31. [3] Zelcer N, Tontonoz P. Liver X receptors as integrators of metabolic and inflammatory signaling. J Clin Invest 2006;116:607–14. [4] Kalaany NY, Mangelsdorf DJ. LXRS and FXR: the yin and yang of cholesterol and fat metabolism. Annu Rev Physiol 2006;68:159–91. [5] Fievet C, Staels B. Liver X receptor modulators: effects on lipid metabolism and potential use in the treatment of atherosclerosis. Biochem Pharmacol 2009;77:1316–27. [6] Desvergne B, Michalik L, Wahli W. Transcriptional regulation of metabolism. Physiol Rev 2006;86:465–514. [7] Han SJ, Lonard DM, O’Malley BW. Multi-modulation of nuclear receptor coactivators through posttranslational modifications. Trends Endocrinol Metab 2009;20:8–15. [8] Teboul M, Enmark E, Li Q, Wikstrom AC, Pelto-Huikko M, Gustafsson JA. OR-1, a member of the nuclear receptor superfamily that interacts with the 9-cisretinoic acid receptor. Proc Natl Acad Sci USA 1995;92:2096–100. [9] Lehmann JM, Kliewer SA, Moore LB, Smith-Oliver TA, Oliver BB, Su JL, et al. Activation of the nuclear receptor LXR by oxysterols defines a new hormone response pathway. J Biol Chem 1997;272:3137–40. [10] Song C, Hiipakka RA, Liao S. Selective activation of liver X receptor alpha by 6alpha-hydroxy bile acids and analogs. Steroids 2000;65:423–7. [11] Janowski BA, Grogan MJ, Jones SA, Wisely GB, Kliewer SA, Corey EJ, et al. Structural requirements of ligands for the oxysterol liver X receptors LXRalpha and LXRbeta. Proc Natl Acad Sci USA 1999;96:266–71. [12] Bjorkhem I. Are side-chain oxidized oxysterols regulators also in vivo? J Lipid Res 2009;50(Suppl.):S213–8. [13] Bjorkhem I, Meaney S, Diczfalusy U. Oxysterols in human circulation: which role do they have? Curr Opin Lipidol 2002;13:247–53. [14] Mitro N, Mak PA, Vargas L, Godio C, Hampton E, Molteni V, et al. The nuclear receptor LXR is a glucose sensor. Nature 2007;445:219–23. [15] Lazar MA, Willson TM. Sweet dreams for LXR. Cell Metab 2007;5:159–61. [16] Denechaud PD, Bossard P, Lobaccaro JM, Millatt L, Staels B, Girard J, et al. ChREBP, but not LXRs, is required for the induction of glucose-regulated genes in mouse liver. J Clin Invest 2008;118:956–64. [17] Oosterveer MH, van Dijk TH, Grefhorst A, Bloks VW, Havinga R, Kuipers F, et al. Lxralpha deficiency hampers the hepatic adaptive response to fasting in mice. J Biol Chem 2008;283:25437–45. [18] Lin J, Yang R, Tarr PT, Wu PH, Handschin C, Li S, et al. Hyperlipidemic effects of dietary saturated fats mediated through PGC-1beta coactivation of SREBP. Cell 2005;120:261–73. [19] Herzog B, Hallberg M, Seth A, Woods A, White R, Parker MG. The nuclear receptor cofactor, receptor-interacting protein 140, is required for the regulation of hepatic lipid and glucose metabolism by liver X receptor. Mol Endocrinol 2007;21:2687–97. [20] Jakobsson T, Venteclef N, Toresson G, Damdimopoulos AE, Ehrlund A, Lou X, et al. GPS2 is required for cholesterol efflux by triggering histone demethylation, LXR recruitment, and coregulator assembly at the ABCG1 locus. Mol Cell 2009;34:510–8.

350

M.H. Oosterveer et al. / Progress in Lipid Research 49 (2010) 343–352

[21] Kim GH, Park K, Yeom SY, Lee KJ, Kim G, Ko J, et al. Characterization of ASC-2 as an antiatherogenic transcriptional coactivator of liver X receptors in macrophages. Mol Endocrinol 2009;23:966–74. [22] Chen M, Bradley MN, Beaven SW, Tontonoz P. Phosphorylation of the liver X receptors. FEBS Lett 2006;580:4835–41. [23] Yamamoto T, Shimano H, Inoue N, Nakagawa Y, Matsuzaka T, Takahashi A, et al. Protein kinase A suppresses sterol regulatory element-binding protein1C expression via phosphorylation of liver X receptor in the liver. J Biol Chem 2007;282:11687–95. [24] Torra IP, Ismaili N, Feig JE, Xu CF, Cavasotto C, Pancratov R, et al. Phosphorylation of liver X receptor alpha selectively regulates target gene expression in macrophages. Mol Cell Biol 2008;28:2626–36. [25] Delvecchio CJ, Capone JP. Protein kinase C alpha modulates liver X receptor alpha transactivation. J Endocrinol 2008;197:121–30. [26] Li X, Zhang S, Blander G, Tse JG, Krieger M, Guarente L. SIRT1 deacetylates and positively regulates the nuclear receptor LXR. Mol Cell 2007;28:91–106. [27] Ghisletti S, Huang W, Ogawa S, Pascual G, Lin ME, Willson TM, et al. Parallel SUMOylation-dependent pathways mediate gene- and signal-specific transrepression by LXRs and PPARgamma. Mol Cell 2007;25:57–70. [28] Lee JH, Park SM, Kim OS, Lee CS, Woo JH, Park SJ, et al. Differential SUMOylation of LXRalpha and LXRbeta mediates transrepression of STAT1 inflammatory signaling in IFN-gamma-stimulated brain astrocytes. Mol Cell 2009;35:806–17. [29] Anthonisen EH, Berven L, Holm S, Nygard M, Nebb HI, Gronning-Wang LM. The nuclear receptor LXR is O-GlcNAc modified in response to glucose. J Biol Chem 2009. [30] Issad T, Kuo M. O-GlcNAc modification of transcription factors, glucose sensing and glucotoxicity. Trends Endocrinol Metab 2008;19:380–9. [31] van Straten EME, Meer Hvan, Huijkman N, van Dijk TH, Baller JF, Verkade HJ, et al. Liver X receptor activation acutely induces lipogenesis, but does not affect plasma lipid response to a high-fat diet in adult mice. Am J Physiol Endocrinol Metab 2009. [32] Yoshikawa T, Shimano H, Yahagi N, Ide T, Amemiya-Kudo M, Matsuzaka T, et al. Polyunsaturated fatty acids suppress sterol regulatory element-binding protein 1c promoter activity by inhibition of liver X receptor (LXR) binding to LXR response elements. J Biol Chem 2002;277:1705–11. [33] Wang J, Einarsson C, Murphy C, Parini P, Bjorkhem I, Gafvels M, et al. Studies on LXR- and FXR-mediated effects on cholesterol homeostasis in normal and cholic acid-depleted mice. J Lipid Res 2006;47:421–30. [34] Tobin KA, Ulven SM, Schuster GU, Steineger HH, Andresen SM, Gustafsson JA, et al. Liver X receptors as insulin-mediating factors in fatty acid and cholesterol biosynthesis. J Biol Chem 2002;277:10691–7. [35] Edwards PA, Kennedy MA, Mak PA. LXRs; oxysterol-activated nuclear receptors that regulate genes controlling lipid homeostasis. Vascul Pharmacol 2002;38:249–56. [36] Miao B, Zondlo S, Gibbs S, Cromley D, Hosagrahara VP, Kirchgessner TG, et al. Raising HDL cholesterol without inducing hepatic steatosis and hypertriglyceridemia by a selective LXR modulator. J Lipid Res 2004;45:1410–7. [37] Levin N, Bischoff ED, Daige CL, Thomas D, Vu CT, Heyman RA, et al. Macrophage liver X receptor is required for antiatherogenic activity of LXR agonists. Arterioscler Thromb Vasc Biol 2005;25:135–42. [38] Joseph SB, McKilligin E, Pei L, Watson MA, Collins AR, Laffitte BA, et al. Synthetic LXR ligand inhibits the development of atherosclerosis in mice. Proc Natl Acad Sci USA 2002;99:7604–9. [39] Bradley MN, Hong C, Chen M, Joseph SB, Wilpitz DC, Wang X, et al. Ligand activation of LXR beta reverses atherosclerosis and cellular cholesterol overload in mice lacking LXR alpha and apoE. J Clin Invest 2007;117:2337–46. [40] Verschuren L, de Vries-van der Weij, Zadelaar S, Kleemann R, Kooistra T. LXR agonist suppresses atherosclerotic lesion growth and promotes lesion regression in apoE*3Leiden mice: time course and mechanisms. J Lipid Res 2009;50:301–11. [41] Wrobel J, Steffan R, Bowen SM, Magolda R, Matelan E, Unwalla R, et al. Indazole-based liver X receptor (LXR) modulators with maintained atherosclerotic lesion reduction activity but diminished stimulation of hepatic triglyceride synthesis. J Med Chem 2008;51:7161–8. [42] Peng D, Hiipakka RA, Dai Q, Guo J, Reardon CA, Getz GS, et al. Antiatherosclerotic effects of a novel synthetic tissue-selective steroidal liver X receptor agonist in low-density lipoprotein receptor-deficient mice. J Pharmacol Exp Ther 2008;327:332–42. [43] Quinet EM, Basso MD, Halpern AR, Yates DW, Sheffan RJ, Clerin V, et al. LXR ligand lowers LDL cholesterol in primates, is lipid neutral in hamster, and reduces atherosclerosis in mouse. J Lipid Res 2009. [44] Kratzer A, Buchebner M, Pfeifer T, Becker TM, Uray G, Miyazaki M, et al. Synthetic LXR agonist attenuates plaque formation in apoE-/- mice without inducing liver steatosis and hypertriglyceridemia. J Lipid Res 2009;50:312–26. [45] Repa JJ, Turley SD, Lobaccaro JA, Medina J, Li L, Lustig K, et al. Regulation of absorption and ABC1-mediated efflux of cholesterol by RXR heterodimers. Science 2000;289:1524–9. [46] Yu L, York J, von BK, Lutjohann D, Cohen JC, Hobbs HH. Stimulation of cholesterol excretion by the liver X receptor agonist requires ATP-binding cassette transporters G5 and G8. J Biol Chem 2003;278:15565–70. [47] Plosch T, Bloks VW, Terasawa Y, Berdy S, Siegler K, Van Der SF, et al. Sitosterolemia in ABC-transporter G5-deficient mice is aggravated on activation of the liver-X receptor. Gastroenterology 2004;126:290–300.

[48] Kruit JK, Plosch T, Havinga R, Boverhof R, Groot PH, Groen AK, et al. Increased fecal neutral sterol loss upon liver X receptor activation is independent of biliary sterol secretion in mice. Gastroenterology 2005;128:147–56. [49] Naik SU, Wang X, Da Silva JS, Jaye M, Macphee CH, Reilly MP, et al. Pharmacological activation of liver X receptors promotes reverse cholesterol transport in vivo. Circulation 2006;113:90–7. [50] van der Veen JN, van Dijk TH, Vrins CL, van Meer H, Havinga R, Bijsterveld K, et al. Activation of the liver X receptor stimulates trans-intestinal excretion of plasma cholesterol. J Biol Chem 2009;284:19211–9. [51] Calpe-Berdiel L, Rotllan N, Fievet C, Roig R, Blanco-Vaca F, Escola-Gil JC. Liver X receptor-mediated activation of reverse cholesterol transport from macrophages to feces in vivo requires ABCG5/G8. J Lipid Res 2008;49:1904–11. [52] Wang N, Silver DL, Costet P, Tall AR. Specific binding of ApoA-I, enhanced cholesterol efflux, and altered plasma membrane morphology in cells expressing ABC1. J Biol Chem 2000;275:33053–8. [53] Wang N, Lan D, Chen W, Matsuura F, Tall AR. ATP-binding cassette transporters G1 and G4 mediate cellular cholesterol efflux to high-density lipoproteins. Proc Natl Acad Sci USA 2004;101:9774–9. [54] Schwartz K, Lawn RM, Wade DP. ABC1 gene expression and ApoA-I-mediated cholesterol efflux are regulated by LXR. Biochem Biophys Res Commun 2000;274:794–802. [55] Larrede S, Quinn CM, Jessup W, Frisdal E, Olivier M, Hsieh V, et al. Stimulation of cholesterol efflux by LXR agonists in cholesterol-loaded human macrophages is ABCA1-dependent but ABCG1-independent. Arterioscler Thromb Vasc Biol 2009;29:1930–6. [56] Venkateswaran A, Laffitte BA, Joseph SB, Mak PA, Wilpitz DC, Edwards PA, et al. Control of cellular cholesterol efflux by the nuclear oxysterol receptor LXR alpha. Proc Natl Acad Sci USA 2000;97:12097–102. [57] Hozoji M, Munehira Y, Ikeda Y, Makishima M, Matsuo M, Kioka N, et al. Direct interaction of nuclear liver X receptor-beta with ABCA1 modulates cholesterol efflux. J Biol Chem 2008;283:30057–63. [58] Timmins JM, Lee JY, Boudyguina E, Kluckman KD, Brunham LR, Mulya A, et al. Targeted inactivation of hepatic Abca1 causes profound hypoalphalipoproteinemia and kidney hypercatabolism of apoA-I. J Clin Invest 2005;115:1333–42. [59] Brunham LR, Kruit JK, Iqbal J, Fievet C, Timmins JM, Pape TD, et al. Intestinal ABCA1 directly contributes to HDL biogenesis in vivo. J Clin Invest 2006;116:1052–62. [60] Brunham LR, Kruit JK, Pape TD, Parks JS, Kuipers F, Hayden MR. Tissue-specific induction of intestinal ABCA1 expression with a liver X receptor agonist raises plasma HDL cholesterol levels. Circ Res 2006;99:672–4. [61] Tang W, Ma Y, Jia L, Ioannou YA, Davies JP, Yu L. Niemann-Pick C1-like 1 is required for an LXR agonist to raise plasma HDL cholesterol in mice. Arterioscler Thromb Vasc Biol 2008;28:448–54. [62] Grefhorst A, Elzinga BM, Voshol PJ, Plosch T, Kok T, Bloks VW, et al. Stimulation of lipogenesis by pharmacological activation of the liver X receptor leads to production of large, triglyceride-rich very low density lipoprotein particles. J Biol Chem 2002;277:34182–90. [63] Cao G, Beyer TP, Yang XP, Schmidt RJ, Zhang Y, Bensch WR, et al. Phospholipid transfer protein is regulated by liver X receptors in vivo. J Biol Chem 2002;277:39561–5. [64] Zhang Y, Repa JJ, Gauthier K, Mangelsdorf DJ. Regulation of lipoprotein lipase by the oxysterol receptors, LXRalpha and LXRbeta. J Biol Chem 2001;276:43018–24. [65] Luo Y, Tall AR. Sterol upregulation of human CETP expression in vitro and in transgenic mice by an LXR element. J Clin Invest 2000;105:513–20. [66] Huuskonen J, Olkkonen VM, Ehnholm C, Metso J, Julkunen I, Jauhiainen M. Phospholipid transfer is a prerequisite for PLTP-mediated HDL conversion. Biochemistry 2000;39:16092–8. [67] Settasatian N, Duong M, Curtiss LK, Ehnholm C, Jauhiainen M, Huuskonen J, et al. The mechanism of the remodeling of high density lipoproteins by phospholipid transfer protein. J Biol Chem 2001;276: 26898–905. [68] Rinninger F, Kaiser T, Mann WA, Meyer N, Greten H, Beisiegel U. Lipoprotein lipase mediates an increase in the selective uptake of high density lipoprotein-associated cholesteryl esters by hepatic cells in culture. J Lipid Res 1998;39:1335–48. [69] Panzenboeck U, Wintersberger A, Levak-Frank S, Zimmermann R, Zechner R, Kostner GM, et al. Implications of endogenous and exogenous lipoprotein lipase for the selective uptake of HDL3-associated cholesteryl esters by mouse peritoneal macrophages. J Lipid Res 1997;38:239–53. [70] Tall AR. Plasma lipid transfer proteins. J Lipid Res 1986;27:361–7. [71] Jiang XC, Beyer TP, Li Z, Liu J, Quan W, Schmidt RJ, et al. Enlargement of high density lipoprotein in mice via liver X receptor activation requires apolipoprotein E and is abolished by cholesteryl ester transfer protein expression. J Biol Chem 2003;278:49072–8. [72] Chiang JY, Kimmel R, Stroup D. Regulation of cholesterol 7alpha-hydroxylase gene (CYP7A1) transcription by the liver orphan receptor (LXRalpha). Gene 2001;262:257–65. [73] Plosch T, Kok T, Bloks VW, Smit MJ, Havinga R, Chimini G, et al. Increased hepatobiliary and fecal cholesterol excretion upon activation of the liver X receptor is independent of ABCA1. J Biol Chem 2002;277:33870–7. [74] Steffensen KR, Gustafsson JA. Putative metabolic effects of the liver X receptor (LXR). Diabetes 2004;53(Suppl. 1):S36–42.

M.H. Oosterveer et al. / Progress in Lipid Research 49 (2010) 343–352 [75] van der Velde A, Vrins CL, van den OK, Kunne C, Oude Elferink RP, Kuipers F, et al. Direct intestinal cholesterol secretion contributes significantly to total fecal neutral sterol excretion in mice. Gastroenterology 2007;133:967–75. [76] van der Velde A, Vrins CL, van den OK, Seemann I, Oude Elferink RP, Van EM, et al. Regulation of direct transintestinal cholesterol excretion in mice. Am J Physiol Gastrointest Liver Physiol 2008;295:G203–8. [77] Brown JM, Bell III TA, Alger HM, Sawyer JK, Smith TL, Shah R, et al. Targeted depletion of hepatic ACAT2-driven cholesterol esterification reveals a nonbiliary route for fecal neutral sterol loss. J Biol Chem 2008;283:10522–34. [78] Zelcer N, Hong C, Boyadjian R, Tontonoz P. LXR regulates cholesterol uptake through Idol-dependent ubiquitination of the LDL receptor. Science 2009;325:100–4. [79] Tabet F, Rye KA. High-density lipoproteins, inflammation and oxidative stress. Clin Sci (Lond) 2009;116:87–98. [80] McGrath KC, Li XH, Puranik R, Liong EC, Tan JT, Dy VM, et al. Role of 3betahydroxysteroid-delta 24 reductase in mediating antiinflammatory effects of high-density lipoproteins in endothelial cells. Arterioscler Thromb Vasc Biol 2009;29:877–82. [81] van der Veen JN, Havinga R, Bloks VW, Groen AK, Kuipers F. Cholesterol feeding strongly reduces hepatic VLDL-triglyceride production in mice lacking the liver X receptor alpha. J Lipid Res 2007;48:337–47. [82] Inaba T, Matsuda M, Shimamura M, Takei N, Terasaka N, Ando Y, et al. Angiopoietin-like protein 3 mediates hypertriglyceridemia induced by the liver X receptor. J Biol Chem 2003;278:21344–51. [83] Shimizugawa T, Ono M, Shimamura M, Yoshida K, Ando Y, Koishi R, et al. ANGPTL3 decreases very low density lipoprotein triglyceride clearance by inhibition of lipoprotein lipase. J Biol Chem 2002;277:33742–8. [84] Zhou J, Febbraio M, Wada T, Zhai Y, Kuruba R, He J, et al. Hepatic fatty acid transporter Cd36 is a common target of LXR, PXR, and PPARgamma in promoting steatosis. Gastroenterology 2008;134:556–67. [85] Shimomura I, Shimano H, Korn BS, Bashmakov Y, Horton JD. Nuclear sterol regulatory element-binding proteins activate genes responsible for the entire program of unsaturated fatty acid biosynthesis in transgenic mouse liver. J Biol Chem 1998;273:35299–306. [86] Liang G, Yang J, Horton JD, Hammer RE, Goldstein JL, Brown MS. Diminished hepatic response to fasting/refeeding and liver X receptor agonists in mice with selective deficiency of sterol regulatory element-binding protein-1c. J Biol Chem 2002;277:9520–8. [87] Repa JJ, Liang G, Ou J, Bashmakov Y, Lobaccaro JM, Shimomura I, et al. LXRalpha and LXRbeta. Genes Dev 2000;14:2819–30. [88] Shimomura I, Bashmakov Y, Ikemoto S, Horton JD, Brown MS, Goldstein JL. Insulin selectively increases SREBP-1c mRNA in the livers of rats with streptozotocin-induced diabetes. Proc Natl Acad Sci USA 1999;96:13656–61. [89] Cha JY, Repa JJ. The liver X receptor (LXR) and hepatic lipogenesis. The carbohydrate-response element-binding protein is a target gene of LXR. J Biol Chem 2007;282:743–51. [90] Chu K, Miyazaki M, Man WC, Ntambi JM. Stearoyl-coenzyme A desaturase 1 deficiency protects against hypertriglyceridemia and increases plasma highdensity lipoprotein cholesterol induced by liver X receptor activation. Mol Cell Biol 2006;26:6786–98. [91] Lund EG, Peterson LB, Adams AD, Lam MH, Burton CA, Chin J, et al. Different roles of liver X receptor alpha and beta in lipid metabolism: effects of an alpha-selective and a dual agonist in mice deficient in each subtype. Biochem Pharmacol 2006;71:453–63. [92] Hellemans KH, Hannaert JC, Denys B, Steffensen KR, Raemdonck C, Martens GA, et al. Susceptibility of pancreatic beta cells to fatty acids is regulated by LXR/PPARalpha-dependent stearoyl-coenzyme A desaturase. PLoS One 2009;4:e7266. [93] Peter A, Weigert C, Staiger H, Rittig K, Cegan A, Lutz P, et al. Induction of stearoyl-CoA desaturase protects human arterial endothelial cells against lipotoxicity. Am J Physiol Endocrinol Metab 2008;295:E339–49. [94] Basciano H, Miller A, Baker C, Naples M, Adeli K. LXRalpha activation perturbs hepatic insulin signaling and stimulates production of apolipoprotein Bcontaining lipoproteins. Am J Physiol Gastrointest Liver Physiol 2009;297:G323–32. [95] Grefhorst A, Parks EJ. Reduced insulin-mediated inhibition of VLDL secretion upon pharmacological activation of the liver X receptor in mice. J Lipid Res 2009;50:1374–83. [96] Masson D, Staels B, Gautier T, Desrumaux C, Athias A, Le GN, et al. Cholesteryl ester transfer protein modulates the effect of liver X receptor agonists on cholesterol transport and excretion in the mouse. J Lipid Res 2004;45:543–50. [97] Briand F, Treguier M, Andre A, Grillot D, Issandou M, Ouguerram K, et al. Liver X receptor activation promotes macrophage-to-feces reverse cholesterol transport in a dyslipidemic hamster model. J Lipid Res 2009. [98] Cao G, Liang Y, Broderick CL, Oldham BA, Beyer TP, Schmidt RJ, et al. Antidiabetic action of a liver X receptor agonist mediated by inhibition of hepatic gluconeogenesis. J Biol Chem 2003;278:1131–6. [99] Laffitte BA, Chao LC, Li J, Walczak R, Hummasti S, Joseph SB, et al. Activation of liver X receptor improves glucose tolerance through coordinate regulation of glucose metabolism in liver and adipose tissue. Proc Natl Acad Sci USA 2003;100:5419–24. [100] Grefhorst A, van Dijk TH, Hammer A, van der Sluijs FH, Havinga R, Havekes LM, et al. Differential effects of pharmacological liver X receptor activation on hepatic and peripheral insulin sensitivity in lean and ob/ob mice. Am J Physiol Endocrinol Metab 2005;289:E829–38.

351

[101] Commerford SR, Vargas L, Dorfman SE, Mitro N, Rocheford EC, Mak PA, et al. Dissection of the insulin-sensitizing effect of liver X receptor ligands. Mol Endocrinol 2007;21:3002–12. [102] Ayala JE, Bracy DP, McGuinness OP, Wasserman DH. Considerations in the design of hyperinsulinemic-euglycemic clamps in the conscious mouse. Diabetes 2006;55:390–7. [103] Gerin I, Dolinsky VW, Shackman JG, Kennedy RT, Chiang SH, Burant CF, et al. LXRbeta is required for adipocyte growth, glucose homeostasis, and beta cell function. J Biol Chem 2005;280:23024–31. [104] Green CD, Jump DB, Olson LK. Elevated insulin secretion from liver X receptor-activated pancreatic beta-cells involves increased de novo lipid synthesis and triacylglyceride turnover. Endocrinology 2009;150:2637–45. [105] Ogihara T, Chuang JC, Vestermark GL, Garmey JC, Ketchum RJ, Huang X, et al. Liver X receptor agonists augment human islet function through activation of anaplerotic pathways and glycerolipid/FFA cycling. J Biol Chem 2009. [106] Korach-Andre M, Parini P, Larsson L, Arner A, Steffensen KR, Gustafsson JA. Separate and overlapping metabolic functions of LXR{alpha} and LXR{beta} in C57Bl/6 female mice. Am J Physiol Endocrinol Metab 2009. [107] Ross SE, Erickson RL, Gerin I, DeRose PM, Bajnok L, Longo KA, et al. Microarray analyses during adipogenesis: understanding the effects of Wnt signaling on adipogenesis and the roles of liver X receptor alpha in adipocyte metabolism. Mol Cell Biol 2002;22:5989–99. [108] Stenson BM, Ryden M, Steffensen KR, Wahlen K, Pettersson AT, Jocken JW, et al. Activation of liver X receptor regulates substrate oxidation in white adipocytes. Endocrinology 2009;150:4104–13. [109] Wang H, Zhang Y, Yehuda-Shnaidman E, Medvedev AV, Kumar N, Daniel KW, et al. Liver X receptor alpha is a transcriptional repressor of the uncoupling protein 1 gene and the brown fat phenotype. Mol Cell Biol 2008;28:2187–200. [110] Kalaany NY, Gauthier KC, Zavacki AM, Mammen PP, Kitazume T, Peterson JA, et al. LXRs regulate the balance between fat storage and oxidation. Cell Metab 2005;1:231–44. [111] Stulnig TM, Steffensen KR, Gao H, Reimers M, Hlman-Wright K, Schuster GU, et al. Novel roles of liver X receptors exposed by gene expression profiling in liver and adipose tissue. Mol Pharmacol 2002;62:1299–305. [112] Steffensen KR, Neo SY, Stulnig TM, Vega VB, Rahman SS, Schuster GU, et al. Genome-wide expression profiling; a panel of mouse tissues discloses novel biological functions of liver X receptors in adrenals. J Mol Endocrinol 2004;33:609–22. [113] Nilsson M, Stulnig TM, Lin CY, Yeo AL, Nowotny P, Liu ET, et al. Liver X receptors regulate adrenal steroidogenesis and hypothalamic–pituitary– adrenal feedback. Mol Endocrinol 2007;21:126–37. [114] Connelly MA. SR-BI-mediated HDL cholesteryl ester delivery in the adrenal gland. Mol Cell Endocrinol 2009;300:83–8. [115] McMahon M, Gerich J, Rizza R. Effects of glucocorticoids on carbohydrate metabolism. Diabetes Metab Rev 1988;4:17–30. [116] Greeve I, Hermans-Borgmeyer I, Brellinger C, Kasper D, Gomez-Isla T, Behl C, et al. The human DIMINUTO/DWARF1 homolog seladin-1 confers resistance to Alzheimer’s disease-associated neurodegeneration and oxidative stress. J Neurosci 2000;20:7345–52. [117] Wang Y, Rogers PM, Stayrook KR, Su C, Varga G, Shen Q, et al. The selective Alzheimer’s disease indicator-1 gene (Seladin-1/DHCR24) is a liver X receptor target gene. Mol Pharmacol 2008;74:1716–21. [118] Koldamova RP, Lefterov IM, Staufenbiel M, Wolfe D, Huang S, Glorioso JC, et al. The liver X receptor ligand T0901317 decreases amyloid beta production in vitro and in a mouse model of Alzheimer’s disease. J Biol Chem 2005;280:4079–88. [119] Zelcer N, Khanlou N, Clare R, Jiang Q, Reed-Geaghan EG, Landreth GE, et al. Attenuation of neuroinflammation and Alzheimer’s disease pathology by liver X receptors. Proc Natl Acad Sci USA 2007;104:10601–6. [120] Riddell DR, Zhou H, Comery TA, Kouranova E, Lo CF, Warwick HK, et al. The LXR agonist TO901317 selectively lowers hippocampal Abeta42 and improves memory in the Tg2576 mouse model of Alzheimer’s disease. Mol Cell Neurosci 2007;34:621–8. [121] Vanmierlo T, Bloks VW, van Vark-van der Zee LC, Rutten K, Kerksiek A, Friedrichs S, et al. Alterations in Brain Cholesterol Metabolism in the APPSLxPS1mut mouse, a Model for Alzheimer’s Disease. J Alzheimers Dis 2009. [122] Vanmierlo T, Rutten K, Dederen J, Bloks VW, van Vark-van der Zee LC, Kuipers F, et al. Liver X receptor activation restores memory in aged AD mice without reducing amyloid. Neurobiol Aging 2009. [123] Mitro N, Vargas L, Romeo R, Koder A, Saez E. T0901317 is a potent PXR ligand: implications for the biology ascribed to LXR. FEBS Lett 2007;581:1721–6. [124] Kumar N, Solt LA, Conkright JJ, Wang Y, Istrate MA, Busby SA, et al. The benzenesulfonamide T0901317 is a novel ROR{alpha}/{gamma} inverse agonist. Mol Pharmacol 2009. [125] Hoekstra M, Lammers B, Out R, Li Z, Van EM, Van Berkel TJ. Activation of the nuclear receptor PXR decreases plasma LDL-cholesterol levels and induces hepatic steatosis in LDL receptor knockout mice. Mol Pharm 2009;6:182–9. [126] de HW, de Vries-van der Weij, Mol IM, Hoekstra M, Romijn JA, Jukema JW, et al. PXR agonism decreases plasma HDL levels in ApoE3-Leiden.CETP mice. Biochim Biophys Acta 2009;1791:191–7. [127] Groot PH, Pearce NJ, Yates JW, Stocker C, Sauermelch C, Doe CP, et al. Synthetic LXR agonists increase LDL in CETP species. J Lipid Res 2005;46:2182–91.

352

M.H. Oosterveer et al. / Progress in Lipid Research 49 (2010) 343–352

[128] Katz A, Udata C, Ott E, Hickey L, Burczynski ME, Burghart P, et al. Safety, pharmacokinetics, and pharmacodynamics of single doses of LXR-623, a novel liver X-receptor agonist, in healthy participants. J Clin Pharmacol 2009;49:643–9. [129] Rader DJ. Liver X receptor and farnesoid X receptor as therapeutic targets. Am J Cardiol 2007;100:n15–9. [130] Zhu Y, Li Y. Liver X receptors as potential therapeutic targets in atherosclerosis. Clin Invest Med 2009;32:E383–94. [131] Colin S, Bourguignon E, Boullay AB, Tousaint JJ, Huet S, Caira F, et al. Intestinespecific regulation of PPARalpha gene transcription by liver X receptors. Endocrinology 2008;149:5128–35. [132] Lalloyer F, Pedersen TA, Gross B, Lestavel S, Yous S, Vallez E, et al. Rexinoid bexarotene modulates triglyceride but not cholesterol metabolism via genespecific permissivity of the RXR/LXR heterodimer in the liver. Arterioscler Thromb Vasc Biol 2009;29:1488–95. [133] Hernandez Vallejo SJ, Alqub M, Luquet S, Cruciani-Guglielmacci C, Delerive P, Lobaccaro JM, et al. Short-term adaptation of postprandial lipoprotein secretion and intestinal gene expression to a high-fat diet. Am J Physiol Gastrointest Liver Physiol 2009;296:G782–92. [134] Albers M, Blume B, Schlueter T, Wright MB, Kober I, Kremoser C, et al. J Biol Chem 2006;281:4920–30. [135] Wagner BL, Valledor AF, Shao G, Daige CL, Bischoff ED, Petrowski M, et al. Promoter-specific roles for liver X receptor/corepressor complexes in the regulation of ABCA1 and SREBP1 gene expression. Mol Cell Biol 2003;23:5780–9.

[136] Lehar J, Krueger AS, Avery W, Heilbut AM, Johansen LM, Price ER, et al. Synergistic drug combinations tend to improve therapeutically relevant selectivity. Nat Biotechnol 2009;27:659–66. [137] Beyer TP, Schmidt RJ, Foxworthy P, Zhang Y, Dai J, Bensch WR, et al. Coadministration of a liver X receptor agonist and a proxisome proliferator activator receptor a agonist in mice-effects of nuclear receptor interplay on high-density lipoprotein and triglyceride metabolism in vivo. J Pharmacol Exp Ther 2004. [138] Oosterveer MH, Grefhorst A, van Dijk TH, Havinga R, Staels B, Kuipers F, et al. Fenofibrate simultaneously induces hepatic fatty acid oxidation, synthesis and elongation in mice. J Biol Chem 2009. [139] Watanabe M, Houten SM, Wang L, Moschetta A, Mangelsdorf DJ, Heyman RA, et al. Bile acids lower triglyceride levels via a pathway involving FXR, SHP, and SREBP-1c. J Clin Invest 2004;113:1408–18. [140] Hellerstein MK. In vivo measurement of fluxes through metabolic pathways: the missing link in functional genomics and pharmaceutical research. Annu Rev Nutr 2003;23:379–402. [141] Hornberg JJ, Bruggeman FJ, Bakker BM, Westerhoff HV. Metabolic control analysis to identify optimal drug targets. Prog Drug Res 2007;64:171, 173– 89. [142] Hellerstein MK. Exploiting complexity and the robustness of network architecture for drug discovery. J Pharmacol Exp Ther 2008;325: 1–9.