regulation of metabolism by nuclear hormone …...this review will be on the function of these...

Regulation of Metabolism byNuclear Hormone Receptors

Huey‐Jing Huang* andIra G. Schulman{

*Department of Biology, Exelixis Inc.,4757 Nexus Centre Drive, San Diego,California 92121{Center for Molecular Design andDepartment of Pharmacology, Universityof Virginia Health System, Charlottesville,Virginia 22908

I. Introduction.... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... 2II. The PPARs ..... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... 4

A. PPARa . .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... 4B. PPARg . .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... 5C. PPARd . .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... 7D. PPARs and Atherosclerosis... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... 9E. PPARs and Inflammation..... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... 10

III. LXR....... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... 12A. Regulation of Hepatic Lipid Metabolism by LXR ...... .. .. ... .. ... .. ... .. .. ... 13B. Regulation of Reverse Cholesterol Transport by LXR ..... ... .. ... .. ... .. .. ... 13C. LXR and Atherosclerosis ..... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... 15D. LXR and Inflammation .... ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... 15E. LXR and Diabetes ...... .. .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... 17F. Therapeutic Potential of LXR Ligands ..... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... 18

IV. FXR....... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... 19A. FXR and the Control of Bile Metabolism....... .. ... .. ... .. .. ... .. ... .. ... .. .. ... 20B. Gallstones, Cholestasis, and Bacterial Growth...... .. ... .. .. ... .. ... .. ... .. .. ... 21C. FXR and Lipid Metabolism....... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... 22D. FXR and Atherosclerosis ..... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... 23E. FXR and Diabetes ...... .. .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... 24F. Control of Liver Regeneration and Tumorigenesis by FXR ...... .. ... .. .. ... 24G. Therapeutic Potential of FXR Ligands ..... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... 25

V. RORa .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... 26A. Regulation of Lipid Metabolism by RORs...... .. ... .. ... .. .. ... .. ... .. ... .. .. ... 26B. Role of RORs in Circadian Rhythm Control and Links to Metabolism.... 28C. RORs and Inflammation...... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... 30D. Therapeutic Potential of RORa Ligands ..... ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... 31

VI. ERRa .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... 31A. ERRa and the PGC‐1a Pathway...... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... 32B. ERRa and Diabetes .... .. .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... 34C. ERRa in Adipose and Intestine .... ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. .. ... 34

Progress in Molecular Biology Copyright 2009, Elsevier Inc.and Translational Science, Vol. 87 1 All rights reserved.DOI: 10.1016/S1877-1173(09)87001-4 1877-1173/09 $35.00

D. ERRa and Cancer.... ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. 35E. Therapeutic Potential of ERRa Ligands ...... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. 36

VII. Summary...... .. ... .. ... .. .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. 37References.... .. ... .. ... .. .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... .. .. ... .. 37

The worldwide epidemic of metabolic disease indicates that a betterunderstanding of the pathways contributing to the pathogenesis of this constel-lation of diseases need to be determined. Nuclear hormone receptors comprisea superfamily of ligand‐activated transcription factors that control develop-ment, differentiation, and metabolism. Over the last 15 years a growingnumber of nuclear receptors have been identified that coordinate geneticnetworks regulating lipid metabolism and energy utilization. Several of thesereceptors directly sample the levels of metabolic intermediates and use thisinformation to regulate the synthesis, transport, and breakdown of the metab-olite of interest. In contrast, other family members sense metabolic activity viathe presence or absence of interacting proteins. The ability of these nuclearreceptors to impact metabolism and inflammation will be discussed and thepotential of each receptor subfamily to serve as drug targets for metabolicdisease will be highlighted.

I. Introduction

There is currently a worldwide epidemic of metabolic disease characterizedby obesity, type II diabetes, hypertension, and cardiovascular disease. Thefactors behind this epidemic appear to be combination of genetic predisposi-tion, high caloric diets, and our increasingly sedentary lifestyles. Indeed recentstatistics from the American Heart Association indicate that almost 50% ofAmerican adults are at risk for cardiovascular disease and the Center forDisease Control reports that approximately 16 million have type II diabetes.Although a number of drugs are currently available to treat the constellation ofmetabolic ailments, the growing epidemic indicates that we still require abetter understanding of the genetic networks and signal transductionsystems that underlie the pathogenesis of these conditions. Further definitionof the factors responsible for metabolic control may pave the way toward newdrug targets with novel mechanisms of action for the treatment of humandisease.

Nuclear receptors comprise a superfamily of ligand‐dependent transcrip-tion factors that regulate genetic networks controlling cell growth, develop-ment, and metabolism. Consisting of 48 members in the human genome thesuperfamily includes the well‐known receptors for steroids, thyroid hormones,

2 HUANG AND SCHULMAN

and vitamins.1 Members of the nuclear receptor superfamily are characterizedby a conserved structural and functional organization consisting of a heteroge-neous amino terminal domain, a highly conserved central DNA‐bindingdomain (DBD), and a functionally complex carboxy terminal ligand‐binding domain (LBD). The LBD mediates ligand binding, receptor homo‐and heterodimerization, repression of transcription in the absence of ligand,and ligand‐dependent activation of transcription when agonist ligands arebound.2

Crystal structures of several LBDs support molecular and biochemicalstudies indicating that ligand binding promotes a conformational change inreceptor structure. What appears to be a relatively flexible conserved helix nearthe carboxy terminus (helix 12) occupies unique positions when structures ofunliganded, agonist‐occupied, and antagonist‐occupied LBDs are compared.3,4

Importantly, mutagenesis experiments indicate that helix 12, referred to asactivation function 2 (AF‐2), is necessary for ligand‐dependent transactivationby nuclear receptors. The AF‐2 helix contributes an essential surface to theformation of an agonist‐dependent hydrophobic pocket that serves as a bindingsite for coactivators. The alternative positions occupied by the helix 12 in theunliganded or antagonist‐occupied conformations preclude the formation ofthis binding pocket.5,6

Classic experiments that defined the effects of glucocorticoids and thyroidhormone on metabolic control provided the foundation for the endocrineregulation of metabolism.7,8 Similar to these classical endocrine receptors, anumber of orphan receptors first cloned based on homology to the well con-served receptor DBD have subsequently been shown to regulate genetic net-works that control important metabolic pathways. In many cases these samepathways are deranged in instances of metabolic disease and it is this class ofmetabolic sensing receptors that will be the focus of this review. Several of thereceptors that will be discussed including the peroxisome proliferator activatedreceptors (PPARs), the liver X receptors (LXRs), and the farnesoid X receptor(FXR) and perhaps the retinoid‐related orphan receptors (RORs) appear tofunction by directly sampling the levels of fatty acids and cholesterol derivativesvia the receptor LBD and regulating genetic networks that control the synthe-sis, transport, and breakdown of the cognate ligand.9–14 Importantly, these fattyacid‐ and cholesterol‐derived natural ligands bind to receptors with affinitiesclose to the physiological concentrations know to exist for these metabo-lites.11,12,15–18 Thus, these receptors are poised to sense and respond to smallchanges in the flux through the metabolic pathways that they control. Theestrogen receptor related receptors (ERRs) comprise an additional subfamilyof nuclear receptors that also appear to play important roles in the regulation ofmetabolism. In contrast to the ligand‐activated receptors mentioned earlier, theactivity of the ERRa in particular appears to be controlled by the presence or

REGULATION OF METABOLISM BY NUCLEAR RECEPTORS 3

absence of interacting proteins instead of lipid‐derived ligands. The focus ofthis review will be on the function of these ‘‘metabolic’’ nuclear receptors, thephysiological pathways they regulate, and their potential as drug targets.

There are several reoccurring themes that appear throughout this review.First, the ability of individual nuclear receptors to regulate multiple geneticnetworks in different tissues has made drug discovery a challenging process forthis class of potential drug targets. Second, in many cases the phenotype of agenetic knockout of a particular nuclear receptor does not accurately predictthe physiological activity of a receptor‐specific small molecule agonist orantagonist. Finally, there is an intimate connection between the regulation ofmetabolism and the control of inflammation.

II. The PPARs

Three distinct members of the PPAR subfamily each encoded by a distinctgene have been identified and well characterized. PPARa (NR1C1) is highlyexpressed in liver, kidney, and muscle. PPARg (NR1C3) is enriched in adiposetissue and PPARb/d (NR1C2; referred to PPARd in this review) appears to beubiquitously expressed. All three PPARs bind to DNA as heterodimers withretinoid X receptors (RXR; NR2B subgroup) and prefer to bind to directrepeats of the nuclear receptor half‐site AGGTCA separated by 1 nucleotide(DR1). Each subtype appears to have unique functions and PPARa and PPARgare the targets of the fibrate and thiazolidinedione (TZD) classes of drugs,respectively.19

A. PPARaPPARa is the molecular target of the fibrate class of drugs used for the

treatment of hypertriglyceridemia. Studies in vitro and in vivo demonstratethat PPARa directly regulates a network of genes encoding the proteinsrequired for the uptake of fatty acids, enzymes required for the oxidation offatty acids (b oxidation), and enzymes required for ketone body utilization bybinding to control regions in the promoter of these genes.19 Thus, activation ofPPARa promotes the utilization of fat as an energy source. Activation of PPARaalso directly induces the genes encoding the apolipoproteins apoAI and apoII,which contribute to the protein core of high density lipoprotein (HDL) parti-cles. Thus, fibrates have the added benefit of slightly raising HDL, the ‘‘goodcholesterol.’’20,21

Given its role in controlling the utilization of fatty acids for energy produc-tion it is not surprising that PPARa is required for the normal response tofasting and starvation. Mice lacking PPARa accumulate triglycerides in the liverand become hypoketonic and hypoglycemic during fasting or starvation.22,23


Recent studies indicate the fibroblast growth factor 21 (FGF21) functions as anendocrine hormone that mediates many of the effects of PPARa. The geneencoding FGF21 is directly induced by PPARa in response to fasting via abinding site in the promoter. FGF21 in turn stimulates lipolysis in adiposetissue and ketogenesis in the liver.24 Taken together PPARa appears to functionas a sensor of the fed/starved state. The increase in fatty acids derived fromadipose during fasting provides PPARa agonists that promote the utilization offat as energy and further stimulates the release of fatty acids from the adiposeby the endocrine action of FGF2124 (Fig. 1).

B. PPARgPPARg is the master transcriptional regulator of adipogenesis and plays an

important role in the process of lipid storage.25 Thus, PPARa and PPARg havecontrasting roles in the regulation of fat metabolism; PPARa promotes theutilization of fat in the liver and muscle while activation of PPARg promotesstorage in adipose. A number of naturally occurring fatty acids and prostanoidshave been shown to act as PPARg agonists; however, perhaps most importantly

Fatty acids

FGF21

Lypolysis

Adipose

Liver

PPARa

b-oxidationketogenesis

FIG. 1. Regulation of fatty acid utilization by PPARa. Fatty acids directly bind to PPARa andact as agonists that increase transcriptional activity. Activated PPARa directly induces genes thatencode the enzymes required for b‐oxidation and ketogenesis. PPARa also increases expression ofthe gene encoding FGF21. FGF21 acts in an autocrine and paracine fashion to further enhanceb‐oxidation and ketogenesis and FGF21 also stimulates lipolysis in adipose tissue to promote therelease of fatty acids. See text for further details.


was the identification that the TZD class of insulin sensitizing drugs includingrosiglitazone (Avandia) and pioglitazone (Actos) are PPARg agonists.26

Although PPARg was identified as the therapeutic target of TZDs in 199526 itis still fair to say that the mechanism underlying the insulin sensitizing activityof this class of drugs is still not completely defined. Fatty acid accumulation ininsulin sensitive tissues such as liver and skeletal muscle has been shown topromote insulin resistance.27 Activation of PPARg in adipose has been pro-posed to increase the number of adipocytes and promote the relocalization andstorage of fat in adipose, protecting peripheral tissues from lipotoxicity.28

Consistent with this idea is the observation that selective knockout of PPARgin adipose eliminates the therapeutic activity of TZDs in mice29 and that acommon side effect of TZD treatment in humans is weight gain due to anincrease in adipose mass.30 PPARg is also expressed at relatively low levels inother tissues and selective knockout in skeletal muscle reduces the therapeuticactivity of TZDs,31 suggesting that there are adipose‐independent sites ofPPARg activity.

The TZDs have proven to be effective drugs for improving insulin sensitiv-ity and treating type II diabetes. However, they are not without problems.Troglitazone, the first TZD in the clinic, was taken off the market because ofcases of drug‐induced liver damage.32 Additionally, recent meta‐analyses haveindicated that treatment with rosiglitazone is associated with increased risk ofmyocardial infarction and deaths due to cardiovascular events.33 Pioglitazonetreatment was also shown to be associated with an increase in serious heartfailure although a significantly lower risk of myocardial infarction and deathwas observed in this patient population.34 Finally, muraglitazar an investiga-tional drug that is a dual agonist of PPARa and PPARg was found to beassociated with an increase in major cardiovascular events and increasedincidence of death.35 The increases in cardiovascular events and mortalityseen with these drugs are relatively small. Nevertheless, the wide scale use ofPPARg agonists in the type II diabetic population has raised serious concernsabout the safety of these drugs for long term therapy.

The molecular basis underlying the increase in cardiovascular events is notclear. Changes in energy metabolism mediated by PPARs could significantlyinfluence cardiac function. Additionally, several studies have identified edemaas a side effect of TZD treatment that could impact heart function.36,37 Regu-lation of the epithelial sodium channel (ENaCg) in the kidney by PPARg hasbeen suggested as potential mechanism behind the TZD‐dependent edema38

but it remains to be seen if inhibiting this channel will decrease the cardiovas-cular events associated with TZD treatment. Based on the recent clinical data itis unlikely that additional PPARg agonists will make it to the clinic unless abetter understanding of the tissue‐specific responses of this receptor isobtained.


C. PPARdNot surprisingly based on its ubiquitous expression pattern, genetic knock-

out of PPARd results in a number abnormalities including embryonic lethalitysecondary to placental defects, decreased adipose mass, mylination deficien-cies, altered inflammatory responses, and impaired wound healing.39 Morerecent studies exploiting additional genetic models and synthetic agonists,however, have uncovered important functions for this receptor in the controlof metabolism and inflammation.39,40

X‐ray crystallography, indicates that PPARd has a relatively large ligandbinding pocket and in vitro studies indicate that fatty acids as well as eicosa-noids including prostaglandin A1 and carbaprostacyclin function as agonists.39

Very low density lipoprotein (VLDL) particle associated fatty acids have alsobeen demonstrated to induce PPARd target genes in a receptor‐dependentmanner41 raising the possibility that PPARd regulates the synthesis, transport,and catabolism of triglyceride‐rich lipoprotein particles. Further support for arole of PPARd in lipoprotein metabolism results from studies exploring theactivity of the PPARd‐specific synthetic agonist GW501516. Treatment ofanimals, including, nonhuman primates with GW501516 significantly increasesHDL particles, lowers triglycerides and low density lipoprotein (LDL) parti-cles, and decreases fasting insulin levels.39,40 Mechanistic studies point toregulation of the gene encoding the ATP binding cassette transporter ABCA1by PPARd as an important step in the control of HDL levels.42 ABCA1 functionsas a cholesterol transporter to transfer cholesterol out of cells toHDLparticles43

and its function will be discussed further in Section III. PPARd mediateddownregulation of intestinal cholesterol absorption via regulation of the geneencoding Niemann‐Pick C1‐like protein 1 (NPCL‐1), a cholesterol transporterthat is the target of the cholesterol lowering drug ezetemide (Zetia), has alsobeen suggested to play a role in the effect of PPARd on lipid levels.44

To examine the role of PPARd in specific tissues, Wang et al.45,46 fused thestrong transcriptional activation domain of the viral transcription factor VP16 tothe amino‐terminus of PPARd to create a ‘‘hyperactive’’ receptor that activatestranscription even in the absence of agonists. Transgenic approaches were thenused to express VP16–PPARd in adipose (white and brown) and skeletalmuscle. In both tissues VP16–PPARd expression produced a dramatic increasein the b‐oxidation of fatty acids. In adipose, the increase fat oxidation led to adecrease in adipose mass and protection from diet‐induced obesity and insulinresistance. The protection against diet‐induced obesity results, at least in part,from increased thermogenesis in brown fat secondary to the induction of genesinvolved in b‐oxidation and the uncoupling of oxidative phosphorylation fromATP production.46 Uncoupling oxidative phosphorylation from ATP productionby expression of uncoupling protein 1 (UCP‐1) leads to a futile cycle that


generates heat when fat is metabolized. Weight loss was also observed in obesemice treated with the synthetic agonist GW501516,45,47 suggesting that thesame PPARd‐dependent pathways can be activated pharmacologically. Never-theless, a significant change in body weight was not detected when obeseRhesus monkeys were treated for 4 months with GW501516.42 As discussedearlier, however, significant agonist‐dependent effects on lipid metabolism andinsulin level was observed in these animals indicating that GW501516 is activein nonhuman primates. Perhaps species‐dependent differences in the bioavail-ability, tissue distribution, and/or efficacy of GW501516 account the differ-ences on adipose mass between rodents and primates.

In skeletal muscle expression of VP16–PPARd induces genes involved inb‐oxidation, mitochondrial respiration, and increases the proportion of slowtwitch oxidative muscle fibers.46 In short the muscle of these animals becomefat burning machines and interestingly these transgenic mice can run on atreadmill for significantly longer times than mice without VP16–PPARd. Giventhe interest in ‘‘performance‐enhancing’’ drugs, Narkar et al.48 tested the abilityof GW501516 to increase endurance in mice (defined as running on a treadmilluntil exhaustion). In contrast to the result observed with the super‐activeVP16–PPARd construct, activation of endogenous PPARd with the syntheticagonist did not increase endurance. When agonist treatment was coupledwith a minimal exercise regimen, however, the combination of drug withexercise produced a significantly larger increase in running time compared toexercise alone. Although pharmacological activation of PPARd alone does notimprove endurance, pharmacologic activation of AMP kinase, a kinase that isactivated when energy levels are low,49 is sufficient by itself to improve endur-ance in sedentary mice.48,50 AMP kinase, like PPARd, is known to play animportant role in muscle fiber type specification50 and the endurance promot-ing activity of an AMP kinase activator is lost in Ppard�/� mice.48 Thus,activation of PPARd is necessary to improve endurance. Interestingly, activatedAMP kinase increases the transcriptional activity of PPARd at least in part byphosphorylation of the peroxisome proliferator activated receptor g coactivator1a (PGC‐1a), a transcriptional coactivator that directly interacts with PPARd.48

AMP kinase activity is also induced by exercise50 suggesting a simple modelthat exercise activated AMP kinase increases the transcriptional activity ofPPARd leading to expression of a genetic network involved in the specificationslow twitch oxidative muscle fibers and improved endurance (Fig. 2A). If thislinear pathway is correct, one must ask why neither exercise alone nor simplyactivating PPARd with a synthetic agonist (bypassing AMP kinase) is sufficientto improve endurance? We would argue that there is a threshold level ofPPARd activity that must be achieved in order to increase endurance (shownschematically in Fig. 2B) and that the exercise regimen used and GW501516


individually do not achieve this level. Perhaps increasing the duration of theexercise or improving the efficacy and bioavailability of the synthetic agonistwould allow these agents to function alone.

Synthetic PPARd ligands have proven to be very effective in preclinicalmodels of diabetes and GW501516 was taken into clinic for the treatment ofdyslipidemia in 2006. It remains to be seen if synthetic ligands for PPARd willprove to be effective for the treatment of human disease.

D. PPARs and AtherosclerosisThe important roles for the PPARs in the control of lipid metabolism

prompted a number of studies investigating the activity of subtype selectiveagonists in mouse models of atherosclerosis.39,51 Generally, LDL receptorknockout (Ldlr�/�) mice or apoE knockout (apoE�/�) mice treated with syn-thetic ligand have been used as model systems. Based on these studies, one canconclude that activation of any of the 3 PPARs reduces atherosclerosis. One,however, could also conclude the opposite; that activation of the PPARs haslittle or no benefit for the treatment of atherosclerosis.51 It is clear that

BA

PP

AR

act

ivity Threshold for

improvedendurance

mRNA

PPARRXR

PGC-1

AMP Kinase

Exercise

PO4

Endurance geneP

PA

R

100

80

60

40

20

0 VP

16–PP

AR

PP

AR

agonist

AM

P agonist

Exercise

Exercise

+P

PA

R agonist

FIG. 2. PPARd‐dependent endurance pathway. (A) Exercise leads to activation of AMP kinasewhich phosphorylates PGC‐1a. Phosphorylated PGC‐1a acts as a transcriptional coactivator toincrease the activity of PPARd leading to the induction of endurance‐promoting genes. (B) Graphillustrates the hypothetical activity of PPARd under different conditions explored by Nakar et al.48

The black line denotes a hypothesized threshold level of PPARd transcriptional activity needed forimproved endurance.


differences in the genetic backgrounds of the mice, their sex, the particularagonist, the diet, and the environment can influence the outcomes of theseexperiments in ways that are not well understood.

Interestingly, treatment of hyperlipidemic Ldlr�/� and apoE�/� mice withPPAR ligands has relatively small effects on plasma lipid levels and little or nochange in total plasma cholesterol is detected even in the studies that reportedagonist‐dependent decreases in atherosclerosis.51 Therefore, the predominanteffect of the PPARs on atherosclerosis does appear to result from changes inplasma lipid levels. A critical event in the development of atherosclerosis is therecruitment of macrophages to the underlying epithelial layer of vessel wallsand the uncontrolled uptake of oxidized cholesterol.52 Continued accumulationof oxidized cholesterol by macrophages and an associated inflammatoryresponse leads to foam cell formation and the initiation of atherosclerosis.52

Importantly, all 3 PPAR subtypes are expressed in macrophages and in vitrostudies indicate that that they can modulate cholesterol homeostasis in thesecells.39,51 The ability of the PPARs to regulate macrophage cholesterol metab-olism lies, at least in part, through their ability to control the expression ofLXRs and subsequent induction of reverse cholesterol transport,53 the processof transporting cholesterol out of cells to HDL (discussed more detail inSection III). PPAR‐dependent/LXR‐independent pathways that modulatemacrophage cholesterol levels have also been identified.54

E. PPARs and InflammationAlong with the accumulation of oxidized LDL, inflammation plays a critical

role in the development and progression of atherosclerosis.52 Additionally, recentfindings indicate a close link between inflammation and insulin resistance.55

Pharmacologic or genetic inhibition of pathways that underlie inflammatoryresponses protect experimental animals from diet‐induced insulin resistance aswell as atherosclerosis,52,56,57 suggesting a direct role of inflammation in thepathology of these diseases. In both diseases, inflammatory responses mediatedby macrophages appear to be crucial for disease progression. In atherosclerosis,cytokines secreted by macrophages at the vessel wall promote the recruitment ofadditional immune cells and the proliferation of smooth muscle cells that con-tribute atherosclerotic lesion development.52 Macrophages also accumulate inthe adipose tissue of obese animals and humanswhere they produce inflammatorymediators that may contribute to the development of insulin resistance.58,59

Anti‐inflammatory activity is a property shared by many members of thenuclear hormone receptor superfamily including the 3 PPAR subtypes andoccurs by inhibition of the transcriptional activity of the proinflammatorytranscription factors activator protein 1 (AP‐1) and nuclear factor kappa B(NFkB).60 A number of mechanisms have been proposed for this process,


termed transrepression, including direct interactions between PPARs and thep65 subunit of NFkB, induction of the inhibitor of kappa B alpha (IkBa),regulation of c‐Jun N‐terminal kinase (JNK) activity, competition for limitingtranscriptional coactivators and corepressors, and inhibition of corepressorclearance from NFkB regulated promoters.60 The contribution of chronicinflammation to metabolic disease has led to the idea that the anti‐inflammatoryactivity of the PPARs, particularly in macrophages, may contribute to thebeneficial effects of PPAR ligands in animal models of atherosclerosis andinsulin resistance.60 To our knowledge this hypothesis has not yet been testedwith either PPAR ligands or PPAR mutants that dissociate the process ofactivation of transcription from the process transrepression. Such dissociatedligands and receptor mutants have been identified for the glucocorticoidreceptor,61,62 suggesting that such reagents could be identified for thePPARs. Nevertheless, recent studies discussed below have indicated thatmacrophages may be critical sites of action for the activity of PPARs.63–66

Resident macrophages in tissues display significant heterogeneity.67

In obesity classically activated macrophages, also called M1 macrophages,accumulate in adipose and play role in mediating an inflammatory responsethat contributes to insulin resistance.68 In lean animals and people, mostadipose associated macrophages display an alternatively activated or M2a phe-notype. Alternatively, activated macrophages are less inflammatory and appearto play roles in tissue repair.68,69 Energy utilization also differs between thesetwo populations of macrophages. Classically activated macrophages predomi-nantly use glucose while a switch to oxidative metabolism is an integral compo-nent of alternative activation; linking metabolic control to macrophagephenotype and inflammation.70 Alternative activation is induced by IL‐4 andIL‐1369 and studies indicating that both PPARg and PPARd are induced in IL‐4/IL‐13 treated macrophages promoted a number of investigators to examine therole of macrophage PPARs in models of diet‐induced obesity.51,63–66,71

Odegaard et al.65 and Hevener et al.63 used selective knockouts and bonemarrow transplantations to delete PPARg in macrophages and observed glu-cose intolerance and increased insulin resistance in mice exposed to a high fatdiet. The Odegaard et al.65 study specifically explored macrophage phenotypeand determined that alternative activation was impaired, suggesting that insulinresistance observed in the absence of PPARg results from increased inflamma-tion from M1 type macrophages. Hevener et al.63 additionally demonstratedthat the therapeutic activity of rosiglitazone was compromised when macro-phage PPARg was selectively eliminated, indicating that the antiinflammatoryactivity of PPARg contributes to the therapeutic activity of TZDs.

Using macrophage selective knockouts similar to those described forPPARg, Kang et al.64 and Odegaard et al.66 demonstrated that PPARd isrequired for turning on the gene expression program corresponding to


alternative activated (M2a) macrophages. The consequences of macrophagePPARd deletion are impaired glucose tolerance and an exacerbation of insulinresistance in response to a high fat diet; once again supporting a role formacrophage inflammation in the pathology of insulin resistance.64,66 Basedon these studies it appears that both PPARg and PPARd play important rolesin establishing the alternative activated phenotype. It has been suggested thatboth receptors must have distinct roles since knockout of either subtype aloneis sufficient to impair alternative activation.66 Nevertheless, the exact functionof each receptor in the alternative activation pathway remains to bedetermined.

The macrophage‐selective knockout experiments described earlier suggestthat the anti‐inflammatory activity of the PPARs may contribute to theirtherapeutic activity. Several investigators have gone a step further and sug-gested that specifically targeting PPARs in macrophages with tissue‐selectivesmall molecules may be a novel and effective method for treating metabolicdisease.63–66,72 The enthusiasm for such approaches, however, must be tem-pered with the realization that other factors including genetic background, diet,and environment may contribute to the knockout phenotypes. In a separatestudy, Marathe et al.73 used bone marrow transplantation approaches to selec-tively knockout PPARg and PPARd in hematopoietic cells either individually ortogether. These authors concluded that in the C58BL/6 mice the two receptorshave little or no impact on the development of diet‐induced obesity and insulinresistance and that rosiglitazone is effective in the absence of macrophagePPARg.73 The roles for PPARs as important regulators of metabolism are welldocumented and agonists for PPARa (fibrates) and PPARg (TZDs) have beenvalidated in humans for the treatment of metabolic disease. Nevertheless, thecontribution of macrophage PPARs to the pathology of metabolic disease andthe beneficial activity of PPAR agonists remains to be determined.

III. LXR

The LXR subgroup of the nuclear receptor superfamily is comprised of twosubtypes, LXRa (NR1H3) and LXRb (NR1H2) that are encoded by separategenes. The founding member of the subgroup LXRa, was originally clonedfrom a liver cDNA library, hence the name liver X receptor, and found to behighly expressed in the liver, kidney, and intestine.74 In contrast, LXRb is moreubiquitously expressed.75 Both LXRs bind to DNA and regulate transcriptionas heterodimers with RXRs with preferred binding to direct repeats of thenuclear receptor half‐site AGGTCA separated by four nucleotides (DR4).74,75


A. Regulation of Hepatic Lipid Metabolism by LXRThe first link between LXR and lipid metabolism came from the identifica-

tion of cholesterol derivatives including 22(R)‐hydroxycholesterol, 24(S)‐hydroxycholesterol, and 24(S),25‐epoxycholesterol as ligands that directlybind to both LXRa and LXRb and increase their transcriptional activity.16–18

More recent studies have also demonstrated that 27‐hydroxycholesterol andcholestenoic acid are LXR ligands.76,77 The identification of hydroxycholester-ols as natural LXR ligands dovetailed nicely with the characterization of LXRaknockout mice. Apparently, normal under standard laboratory conditions,when challenged with a diet rich in cholesterol Lxra�/� mice accumulatemassive amounts of cholesterol in the liver. Molecular analysis uncoveredaberrant regulation of several genes involved in lipid and cholesterol metabo-lism including Cyp7a1, which encodes cholesterol 7a hydroxylase, the rate‐limiting enzyme in the conversion of cholesterol to bile acids.78 Subsequently,the ATP binding cassette transporters ABCG5 and ABCG8 which move cho-lesterol out of the liver and into the intestine were identified as LXR targetgenes.79,80 Thus, an increase in cholesterol levels is predicted to lead to anelevation in the concentration of cholesterol‐derived LXR ligands resulting inthe catabolism of cholesterol to bile acid and the excretion of cholesterol out ofthe liver (Fig. 3). Importantly, Cyp7a1, ABCG5, and ABCG8 all appear to bedirectly regulated by LXR18,79 although the binding site for LXR present in themurine Cyp7a1 gene is not conserved in the human gene.18

Along with effects on cholesterol metabolism activation of LXR agonistsalso increases expression of genes involved in fatty acid metabolism includingthe master transcriptional regulator of fatty acid synthesis, the sterol responseelement binding protein 1c (SREBP1c)81,82 (Fig. 3). Additionally, several of thegenes encoding the enzymes involved in fatty acid metabolism including fattyacid synthase (FAS) and stearoyl CoA desaturase 1 (SCD‐1) are regulateddirectly or indirectly by LXR.83–85 The coordinate upregulation of fatty acidsynthesis with reverse cholesterol transport is most likely to provide lipids forthe transport and storage of cholesterol.

B. Regulation of Reverse Cholesterol Transport by LXRBased on the defined role for LXR in hepatic cholesterol catabolism and

excretion one might have expected that synthetic LXR agonists would lowerplasma cholesterol levels. Quite surprisingly, however, treatment of animalswith LXR agonists significantly elevates HDL cholesterol.82 Gene expressionanalysis in the livers and intestines of LXR agonist‐treated mice identifiedABCA1 as a direct LXR gene and this discovery stimulated great interest inthe therapeutic potential of LXR agonists given the links between ABCA1,HDL metabolism, and atherosclerosis.86–88 ABCA1 is required for the process


of reverse cholesterol transport whereby cells efflux internal cholesterol toacceptor proteins on pre‐b‐HDL particles. Loss of functional ABCA1 resultsin Tangier disease, a condition in which patients have extremely low levels ofcirculating HDL and an increased risk for developing atherosclerosis. Exami-nation of fibroblasts isolated from subjects with Tangier disease reveals thatABCA1 defective cells are unable to efflux cholesterol, suggesting that the lowHDL levels and increased risk of atherosclerosis results from a loss of reversecholesterol transport. Historically, Tangier disease patients present with largeaccumulations of cholesterol‐laden macrophages in their lymph tissues, high-lighting the role of ABCA1 and reverse cholesterol transport in macrophagecholesterol homeostasis.43,89,90 As described in Section II.D, accumulation ofoxidized LDL cholesterol by macrophages in the arterial wall is an initiatingstep in the development of atherosclerotic lesions52 and recent studies withmouse knockouts of ABCA1 further support a link between reverse cholesteroltransport and atherosclerosis.91–93 In support of the role of LXR as a directregulator of ABCA1 expression and activity, treatment of primary macrophages

LXR

LXR

LXR

ABCA1ABCG1

HDL

Macrophage

ABCG5ABCG8

Bile acids

Triglyceride

CholesterolCyp7A

SREBP1c

FIG. 3. Regulation of cholesterol metabolism by LXR. Activation of LXR in macrophagesresults in the upregulation of genes encoding proteins that participate in reverse cholesteroltransport including ABCA1, ABCG1, ABCG4, and apoE. In the liver, LXR activation results inupregulation of the genes encoding cholesterol 7 hydroxylase (Cyp7a), the rate limiting enzyme inthe catabolism of cholesterol to bile acids, and the half ABC transporter ABCG5 and ABCG8 thatfunction together to excrete cholesterol from the liver into the intestine. Thus, activation of LXRincreases the net flux of cholesterol out of the body. LXR also regulates expression of SREBP1c,a master transcriptional regulator of genes involved in fatty acid and triglyceride synthesis. See textfor details.


or cell lines with LXR agonists results in induction of the ABCA1 gene, increaselevels of ABCA1 protein, and an increase in cholesterol efflux.86,88,94 A bindingsite for LXR–RXR heterodimers in the ABCA1 promoter has also beendescribed.86 Subsequent studies identified other proteins involved in the re-verse cholesterol transport including ABCG1, ABCG4, and apoE as direct LXRtarget genes.94–97 Thus, activation of LXR results in the mobilization of choles-terol in the periphery and stimulates the catabolism and excretion of cholesterolwhen it arrives in the liver (Fig. 3). Interestingly, genetic deletion of LXR activityin mice (Lxra�/�/Lxrb�/�) results in the accumulation of cholesterol‐ladenmacrophages and splenomegaly similar to that observed in Tangier diseasepatients.98,99

C. LXR and AtherosclerosisThe accumulation of oxidized LDL cholesterol by macrophages in blood

vessel walls is an early event in the pathogenesis of atherosclerosis and it hadlong been suggested that reversing this process by pumping cholesterol out ofmacrophage foam cells would have an inhibitory effect on the progression ofatherosclerosis.52 The ability of LXR to directly regulate reverse cholesteroltransport in macrophages allowed two experiments to be carried out to test thishypothesis. First, transplantation of lethally irradiated apoE�/� and Ldlr�/�

mice with bone marrow from wild type or Lxra�/�/Lxrb�/� mice demonstratedthat genetic deletion of LXR leads to an increase in atherosclerosis is these wellestablished mouse models.99 Second, treatment of apoE�/� and Ldlr�/�micewith synthetic LXR agonists leads to a reduction in atherosclerosis.100,101

Together the combination of genetic analysis and pharmacology clearly demon-strated the antiatherogenic activity of LXR. Not surprisingly, the mRNA levelsfor LXR target genes including ABCA1 and apoE are elevated in the athero-sclerotic lesions of mice treated with LXR agonists.101 Subsequent studiescombining bone marrow transplantation with the administration of syntheticLXR agonists have demonstrated that LXR activity in macrophages is necessaryfor the antiatherogenic effect of LXR ligands.102

D. LXR and InflammationIt is easy to assume that the ability of LXR to regulate lipid metabolism and

reverse cholesterol transport provides the mechanistic basis for the antiathero-genic activity of LXR.99–101 Experiments using culturedmacrophages, however,also demonstrate that LXR agonists can inhibit the expression of severalproinflammatory genes including iNOS, COX‐2, and MMP‐9 and these com-pounds are effective in a murine model of irritant contact dermatitis.103,104


Molecular studies indicate that activation of LXR decreases the transcriptionalactivity of NFkB using many of the same mechanisms described for the PPARs(see Section II.E).104

Since atherosclerosis is considered an inflammatory disease52 the questionremains whether LXR mediates its antiatherogenic activity via control ofreverse cholesterol transport, by limiting the inflammatory response, or both.Future studies that combine genetically altered macrophages (i.e., Abca1�/�)introduced by bone marrow transplantation along with the administration ofLXR agonists can be used to define the individual contributions of reversecholesterol transport and anti‐inflammatory activity to therapeutic effects ofLXR ligands. Additionally, studies with the glucocorticoid receptor have shownthat it is possible to identify nuclear receptor ligands that repress inflammatorygenes but do not activate positively regulated glucocorticoid receptor targetgenes.61,62 One expects that such dissociated ligands will also be identifiedfor LXR.

A number of studies have suggested a link between viral or bacterialinfections and atherosclerosis.105 In support of this hypothesis enhancedexpression of Toll‐like receptors (TLR), which mediate the innate immuneresponse to invading pathogens via stimulation of proinflammatory pathways,has been detected in human atherosclerotic lesions.106 Interestingly, studies byCastrillo et al.107 indicate that activation of TLR4 inhibits the transcriptionalactivity of LXR and the ability of macrophages to efflux cholesterol. The crosstalk between TLR4 signaling and LXR activity suggests one potential mecha-nistic basis for the impact of infectious agents on cardiovascular disease.

Along with effects in macrophages, recent studies have identified animportant and specific role for LXRb in T cell proliferation.108 T cell activationtriggers induction of the oxysterol‐metabolizing enzyme SULT2B1 and sup-pression of reverse cholesterol transport by decreasing the availability ofendogenous LXR agonists. Consistent with a role for LXRs in T cell prolifera-tion is the observation that knockout of LXRb confers a proliferative advantagewhile binding of agonists to LXRb during T cell activation inhibits mitogen‐driven expansion. LXRa is not expressed in lymphocytes indicating that thisactivity is subtype selective.108 In a coordinate fashion, the SREBP2 dependentpathway for cholesterol synthesis is activated108 (Fig. 4). SREBP2 is the majortranscriptional regulator of proteins required for cholesterol uptake and syn-thesis including the LDL receptor and HMG‐CoA reductase, the rate limitingenzyme in cholesterol biosynthesis.109 This coupling of decreased LXRbactivity with activation SREBP2 insures that intracellular cholesterol levelsare sufficient to support cell growth and the rapid expansion of the T cellpopulation upon stimulation. Interestingly, inactivation of the cholesteroltransporter ABCG1 attenuates the ability of LXR agonists to inhibit prolifera-tion, directly linking cholesterol homeostasis to the antiproliferative action


of LXRb.108 Mice lacking LXRb also exhibit lymphoid hyperplasia and en-hanced responses to antigenic challenge, indicating that proper regulation ofLXR‐dependent sterol metabolism is important for immune responses.108

These results implicate LXRb signaling in a metabolic checkpoint that mod-ulates cell proliferation and immunity.

E. LXR and DiabetesTreatment of experimental animals with LXR agonists leads to increases in

hepatic fatty acid synthesis and plasma triglyceride levels.81,82 Since elevationsin fatty acids have been linked to insulin resistance and type II diabetes severalinvestigators have examined the cross talk between LXR activity and glucosemetabolism. Interestingly, along with upregulating fatty acid synthesis, activa-tion of LXR also represses expression of the genes encoding the enzymesof gluconeogenesis in the liver including phosphoenolpyruvate carboxy kinase(PEPCK) and glucose 6‐phosphatase110,111 and induces expression of GLUT4in adipose tissue.110 Thus, in many ways activation of LXR mimics treatmentwith insulin. Perhaps not surprisingly in light of this ‘‘insulin‐like’’ activity, LXRligands decrease hepatic glucose output and lower blood glucose levels inanimal models of type II diabetes.110,111 The observation, however, that LXR

T cell receptor

SULT2B1

Oxysterols

Reverse cholesterol transportABCG1

LXRb

SREBP2

Cholesterol

Cholesterol

Cholesterol synthesisHMG-CoA reductase

FIG. 4. Regulation of Tcell proliferation by LXRb. Stimulation of T cell proliferation increasesthe expression of SULT2B1 an enzyme that catabolizes oxysterols, the endogenous ligands for LXR.The decrease in LXR ligands reduces the transcription activity of LXRb resulting in a down-regulation of cholesterol leaving cells via the reverse cholesterol transport pathway. In a coordinatefashion, SREBP2 and cholesterol biosynthetic enzymes are upregulated providing cholesterol forcell growth. See text for details.


ligands can behave as insulin sensitizers even in face of relatively largeincreases in plasma triglyceride levels suggests the possibility of a broaderrole for LXR in regulating glucose homeostasis. Indeed, Mitro et al.112 havedemonstrated that glucose can directly bind to LXRs with relatively weakaffinity (millimolar) and function as an agonist. Thus, LXRs may directlysense glucose and regulate carbohydrate levels in manner similar to the controlof cholesterol metabolism. The observation that LXRs are also active in skeletalmuscle113 and that Lxra�/�/Lxrb�/� mice are resistant to diet‐inducedobesity114 further supports a role for the LXRs as important coordinators ofenergy metabolism.

F. Therapeutic Potential of LXR LigandsThe antiatherogenic, anti‐inflammatory, and antidiabetic activities of LXR

agonists in animal models have highlighted the therapeutic potential of LXRaand LXRb. Nevertheless, the link between LXR activity and triglyceride me-tabolism has clearly dampened the enthusiasm surrounding this target class.Treatment of mice and hamsters with synthetic LXR agonists results in asignificant increase in plasma triglyceride levels82 and elevations in LDLcholesterol have been observed in nonhuman primates.115 Treatment ofpatients with a drug that raises lipids is not a viable option for the treatmentof metabolic diseases and approaches to separate the beneficial activities ofLXR ligands from unwanted side effects need to be explored. Furthermore,studies in human cells have shown that LXR agonists also increase expressionof the gene encoding the cholesterol ester transfer protein (CETP).116 CETPfunctions to transfer cholesterol esters from HDL to apolipoprotein B contain-ing lipoprotein particles and CETP activity has been shown to inversely corre-late with atherosclerosis.117–119 Indeed, CETP inhibitors are currently beingexplored for the treatment of atherosclerosis.118

Interestingly, defects in hepatic cholesterol metabolism are detected inLxra�/� single knockout mice indicating that LXRb is not functionally redun-dant with LXRa.78 In contrast, cholesterol and triglyceride levels appear nor-mal in Lxrb�/� mice suggesting that LXRa mediates most, if not all, of theeffects of LXR ligands on triglyceride metabolism.120 The relatively low level ofLXRb in the liver most likely accounts for lack of functional redundancy in thistissue. Nevertheless, in macrophages either LXRa or LXRb alone appears to besufficient to mediate the effects of LXR ligands on reverse cholesterol transportand inflammatory gene expression. Additionally, a recent study by Bradleyet al.121 indicates that the antiatherogenic activity of LXR agonists are main-tained in apoE/�/Lxra�/� double knockout mice, suggesting that LXRb alone issufficient to limit atherosclerosis. Taken together these observations have ledseveral investigators to suggest that LXRb‐selective ligands may provide a


mechanistic basis for identification of LXR ligands with improved therapeuticprofiles.122 The enthusiasm for LXRb‐selective ligands must be tempered withthe realization that the spectrum of activities measured in the complete ab-sence of LXRa activity may differ when a subtype selective synthetic ligand isused. Additionally, the observation that the ligand binding pockets of LXRa andLXRb defined by crystallography123–126 differ by only one amino acid suggeststhat identification of selective ligands may not be simple.

While the therapeutic activity of LXRb‐selective ligands is still an openquestion, it has been possible to identify ligands for other nuclear receptorsthat exhibit a restricted set of activities and therefore allow the separation ofbeneficial therapeutic activities from unwanted side effects. Perhaps the bestexamples of such compounds are the selective estrogen receptor modulatorssuch as roloxifene that function as estrogen receptor agonists in some tissuesand estrogen receptor antagonists in others.127 More recently, synthetic ligandsfor PPARg have been identified that appear to separate the insulin sensitizingactivity of PPARg from unwanted effects on weight gain.128,129 A commonfeature of all these selective receptor modulators is that they appear tofunction as partial or weak agonists when characterized in vitro. When boundto receptors selective modulators produce unique conformational changesthat cannot be achieved by more typical agonists.3,4 The outcome of theseunique conformations is an alteration in interactions between receptors and thedown‐stream coregulator proteins that mediate the transcriptional responseleading to ligand‐specific effects on gene expression.127,130 Since the LXRsfunction in multiple tissues to mediate effects on lipid metabolism, glucosehomeostasis, and inflammation we expect that the identification of selectiveLXR modulators will yield compounds with beneficial therapeutic activities.

IV. FXR

FXRa (NR1H4), like the PPARs and LXRs, was originally identified as anorphan member of the nuclear hormone receptor superfamily and subsequent-ly shown be activated by the direct binding of farnesol.131 FXR binds to DNAas an obligate heterodimer with RXR and prefers to bind to inverted repeats ofthe nuclear receptor half‐site AGGTCA separated by four basepairs(IR4).132,133 A second FXR subtype, FXRb (NR1H5), has been identified inrodents, rabbits, and dogs but is a pseudogene in humans and primates.133

FXRb will not be discussed in this review and we will refer to FXRa as simplyFXR. The expression pattern of FXR is relatively restricted to the liver, intes-tine, kidney, and adrenal gland. Low levels of expression of the FXR mRNAhave also been reported in adipose tissue, heart, and smooth muscle cells,however, it is not clear if FXR is functionally active in the latter three


locations.133 Hepatic mRNA levels of FXR are elevated by prolonged fastingand by overexpression of the transcriptional coactivator PGC‐1a.134 PGC‐1a isan important transcriptional regulator of hepatic gluconeogenesis and energymetabolism135 suggesting that FXR may play a role in the response to nutri-tional status.

A. FXR and the Control of Bile MetabolismA number of studies have demonstrated that bile acids such as chenodeoxy-

cholic acid (CDCA), deoxycholic acid (DCA), and cholic acid (CA) binddirectly to the FXR and function as activators of FXR‐regulated genes.136–138

Importantly, FXR activation by bile acids leads to downregulation of Cyp7a1,the gene encoding the rate limiting enzyme (cholesterol 7a‐hydroxylase) in theconversion of cholesterol to bile acids.136–138 Inhibition of Cyp7a1 by FXRoccurs indirectly via the FXR‐dependent upregulation of the small heterodi-meric partner (SHP, NR0B2) a transcriptional repressor139 (Fig. 5). SHP lacksthe canonical DBD found in most other members of the nuclear receptorsuperfamily but can dimerize with other receptors via a LBD. SHP interactsstrongly with corepressors proteins so dimerization with SHP generally leads to

Liver

FXR

SHP

CYP8B1

CYP7A1

AKR1D1Intestine

Cholesterol

CA MCACDCA

7a -hydroxycholesterol

FGF19 FGF19

FXR

Bile acidresin

FIG. 5. Regulation of bile acid synthesis by FXR. In the liver, bile acids act as FXR agonistsleading to the induction of SHP, a transcriptional repressor. SHP strongly represses the transcrip-tion of CYP8B1 and relatively weakly represses CYP7A1. The net result of activating FXR in theliver is a change in bile acid composition toward the more hydrophobic bile acids MCA (mice) orCDCA (humans). MCA and CDCA poorly promote the absorption of cholesterol in the intestinerelative to CA. In the intestine, activation of FXR increases the expression of FGF19 which acts in aparacine fashion to strongly inhibit CYP7A1 in the liver and an overall decrease in bile acidsynthesis. Bile acids resins sequester bile acids in the intestine, decreasing FXR activity, reducingthe levels of FGF19, and consequently upregulating the levels of CYP7A1. See text for details.


an inhibition of transcription.140 In the context of Cyp7a1, SHP dimerizes withand inhibits the liver receptor homolog 1 (LRH1, NR5A2), a third nuclearhormone receptor that functions as a constitutive activator of Cyp7a1.139 SHPalso inhibits expression of the Cyp8b1 gene encoding sterol 12a‐hydroxy-lase.141,142 Sterol 12a‐hydroxylase sits at a branch point in the bile acid syn-thetic pathway that determines the polarity of primary bile acids (Fig. 5). WhenCyp8b1 is active CA is the primary product; when Cyp8b1 is inactive CDCA(humans) and muricholic acid (MCA) (mice) are the predominant species.143

Along with regulation of bile acid synthetic enzymes via control of SHPexpression, FXR induces the gene encoding FGF19 (FGF15 is the mouseortholog) which also functions to inhibit expression of Cyp7a1144,145 (Fig. 5).FXR19 is expressed in the intestine, not the liver, suggesting that intestinalFXR senses the level of bile acids at this site and regulates hepatic bile acidsynthesis in an endocrine fashion via the production of FGF19.144,145 FGF19binds to FGF receptor 4 on the surface of hepatocytes and appears to inhibitCyp7a1 by activating a JNK‐dependent145 or mitogen activated kinase (MAP)‐dependent146 phosphorylation cascade. FGF19 also stimulates the normalrefilling of the gallbladder with bile acids after cholecystokinin‐dependentemptying.147 Taken together these results suggest that when bile acid levelsare high in the intestine that FXR functions to inhibit further synthesis andstimulate gallbladder filling. Interestingly, tissue‐specific knockouts of FXRindicate that the intestine is the dominant site for FXR‐dependent regulationof Cyp7a1 while the liver is the dominant site for regulation of Cyp8b1.148

Thus, activation of FXR in the intestine leads to an inhibition of bile acidsynthesis while in the liver activation of FXR would primarily impact thecomposition of bile acids produced (Fig. 5). The consequence of altering bileacid composition on lipid metabolism will be discussed further below.

B. Gallstones, Cholestasis, and Bacterial GrowthBile comprised of bile acids, phospholipids, and cholesterol is stored in the

gallbladder and released upon feeding. An excess of cholesterol to bile acids inthe bile leads to the precipitation of cholesterol and formation of cholesterolgallstones. According to the American Gastroenterological Association, almost24 million Americans suffer from gallstones and the condition can be quitepainful. Along with controlling bile acid synthesis, FXR also regulates genesrequired for the hepatic uptake, conjugation, and excretion of bile acids.149–152

The bile salt export pump (BSEP), multidrug resistant‐associated protein 2(MRP2, ABCC2), and the multidrug resistance P‐glycoportein 3 (MDR3,ABCB4) are localized on the canalicular membrane of hepatocytes andsecrete bile acids from hepatocytes into the bile canaliculi.133 These threetransporters are regulated directly by FXR and induced by FXR agonists.132,133

Moschetta et al.153 demonstrated that treatment with the synthetic FXR agonist


GW4064 reduces the number of gallstones in a gallstone susceptible strain ofmice. The beneficial effect of the FXR agonist most likely arises from increasedexpression of the canalicular bile acid transporters and increased transport ofbile acids into the gallbladder. In contrast, FXR knockout mice are moresusceptible to developing gallstones than FXRþ/þ mice when placed on alithogenic diet.153 The current treatment for gallstones is surgical removal ofthe gallbladder (cholecystectomy) raising the possibility that FXR agonistscould provide a novel nonsurgical treatment for this disease.

Cholestasis is defined as any condition that impairs the flow of bile acids outof the liver and is many times associated with liver damage. Generally, chole-static diseases are defined as obstructive (the flow of bile is physically blocked)or nonobstructive.154,155 Not surprisingly, GW4064 and 6‐ethyl CDCA, aderivative of CDCA that is a potent FXR agonist, has proven effective inseveral models of drug induced nonobstructive cholestasis.156–158 The activityof FXR agonists in these models most likely arises from their ability to inducethe transport of bile acids out of the liver and to reduce further bile acidsynthesis. Inflammation and fibrosis are commonly associated with liver dam-age and FXR agonists also reduce the expression of markers of fibrosis andinflammation perhaps via induction of the transcriptional repressor SHP asdescribed for Cyp7a1.159–161 As we have seen with the PPARs and LXRs,regulation of metabolism by FXR appears to be intimately linked with thecontrol of inflammation.

Obstruction of bile flow is also associated with intestinal bacterial growthand injury to the intestinal mucosa. Fxr�/� mice have bacterial overgrowthin the ileum and compromised epithelial barrier most likely resulting fromimpaired bile flow. On the other hand, FXR activation induces a number ofgenes in the intestine involved in enteroprotection including angiogenin, nitricoxide synthase, and IL‐18.162

C. FXR and Lipid MetabolismClinical trials examining the utility of bile acids for the treatment of

cholesterol gallstones first demonstrated that increasing bile acid levels leadsto a corresponding decrease in plasma triglycerides.163–165 The observation thatFxr�/� mice have elevated triglyceride levels166 and that a synthetic FXRagonist lowers triglycerides in rats167 supports an essential role for FXR inmediating the effects of bile acids on triglyceride metabolism. Indeed, FXR hasbeen identified as a direct regulator of a number of genes encoding proteinsinvolved in triglyceride synthesis and catabolism including apoCII, apoCIII,apoAV, FGF19, SREBP1c, syndecan 1, the VLDL receptor, complementC3/acylation stimulating protein, Insig‐2, and PPARa.144,150,151,168–172 Thecontribution of these different mechanisms to FXR‐dependent control oftriglycerides remains to be determined.


While a role for FXR in the control of triglyceride metabolism appears to bewell established, the effect of FXR on cholesterol homeostasis is less clear.Repression of Cyp7a1 by FXR should inhibit the conversion of cholesterol tobile acids resulting in an increase in cholesterol levels (Fig. 5). In support of thisprediction, humans with a genetic deficiency in CYP7A1 present with elevatedcholesterol levels.173 Nevertheless, no evidence for increased cholesterol wasobserved in humans treated with bile acids165 and genetic knockout of FXRresults in an increase in cholesterol levels174,175; not the expected decreasepredicted if feedback inhibition of Cyp7a1 is eliminated. Similarly, studies inanimals and humans have shown that bile acids repress the production ofVLDL.176,177 As described earlier, activation of FXR in the liver will stronglyinhibit Cyp8b1 expression.141,142 Sterol 12a‐hydroxylase, the enzyme encodedby Cyp8b1, sits at a branch point in the bile acid synthetic pathway and itsenzymatic activity is required for the synthesis of CA. The parallel arm in thepathway leads to synthesis of MCA in mice and CDCA in humans.143 Thus,modulating expression of Cyp8b1 expression alters bile composition (Fig. 5).Importantly, individual bile acids differ in their ability to promote intestinalcholesterol absorption and MCA, of all bile acids tested, promotes the lowestamount of cholesterol absorption while CA promotes the greatest amount.178

CDCA is in between CA andMCA 178. Thus, activating FXR in the liver shoulddecrease cholesterol absorption in the intestine. On the other hand, eliminatingFXR activity in the liver with antagonists or by genetic knockout shouldincrease cholesterol absorption by the intestine (Fig. 5). Thus, we wouldargue that many of the effects of modulating FXR activity on lipid levels mayresult from influencing the absorption of fat and cholesterol in the intestine.

D. FXR and AtherosclerosisThe ability of FXR to control triglyceride and cholesterol metabolism

suggests that activating or inhibiting this receptor should have significanteffects on the development and progression of atherosclerosis. Indeed,treatment of apoE�/� mice with 6‐ethyl CDCA, a derivative of CDCA thatfunctions as an FXR agonist, reduces atherosclerosis.179 Based on this data andon studies demonstrating elevated triglyceride and cholesterol levels in Fxr�/�

mice, one would predict that eliminating FXR activity in a proatheroscleroticmouse background would increase atherosclerosis. The predicted result ofincreased atherosclerosis in apoE�/�/Fxr�/� mice was observed in one pub-lished study.174 Two other studies, however, one using apoE�/�/Fxr�/�180 miceand the other using Ldlr�/�/Fxr�/�181 mice detected decreased atherosclerosiscompared to apoE�/� and Ldlr�/� controls. Interestingly, both of the studiesthat measured decreased atherosclerosis detected changes in plasma lipidlevels that one would expect to actually promote atherosclerosis.180,181 Bothstudies also detected decreased expression of CD36 in macrophages isolated


from double knockout mice. CD36 is a scavenger receptor responsible for theuptake of oxidized cholesterol by macrophages and decreased expressionwould be expected to reduce atherosclerosis by limiting macrophage foamcell development.182 FXR is not expressed in macrophages and both studiessuggest that alterations in lipid profiles that result from deleting FXR mayinfluence the expression of CD36 in peripheral cells.180,181 In the study usingthe Ldlr�/� background, the authors also detected a significant increase inVLDL cholesterol and decreases in LDL and HDL.181 This change in lipopro-tein particle profile, particularly the decrease in LDL, may also contribute tothe decrease in atherosclerosis observed in this study. The different resultsseen among the various atherosclerosis studies again highlight the care thatneeds to be taken in interpreting such experiments. Furthermore, these studiesclearly illustrate that it can be difficult to predict the activity of a nuclearreceptor agonist or antagonist from the phenotype of a knockout.

E. FXR and DiabetesAnalysis of Fxr�/� mice has demonstrated impaired glucose tolerance and

insulin resistance in the liver and in peripheral tissues (muscle and fat).183,184

The effects on insulin resistance most likely arise from the elevated fatty acidlevels observed in these mice. Similarly, the synthetic FXR agonist GW4064improves insulin sensitivity in several diabetic models including db/db, ob/ob,and KK‐A(y) mice.185 Infection of these mice with an adenovirus expressinga ‘‘super‐active’’ VP16–FXR construct also improves insulin sensitivity suggest-ing that the liver is the major site of FXR activity with regards to diabetes.185

Examination of these diabetic models suggests that activation of FXR reduceshepatic gluconeogenesis and increases glycogen synthesis thus reducing plas-ma glucose levels.183–185 The effects of FXR on gluconeogenesis and glycogensynthesis are somewhat surprising given the report that FXR is induced byprolonged fasting,134 a condition when gluconeogenesis would be expected tobe high and glycogen synthesis low. The mechanistic basis for these effectsremains to be determined.

F. Control of Liver Regeneration and Tumorigenesisby FXRRecent studies indicate that deletion of FXR in mice impairs the process

the liver regeneration after hepatectomy186 and increases the incidence ofhepatocellular adenoma, carcinoma, and hepatocholangiocellular carcinomain older (12 months) animals.187,188 Opposite results are observed when wild‐type mice are fed diets enriched in bile acids; 187,188 however, to our knowledge


synthetic agonists have not been examined in these models. It appears that theability of FXR to control genes involved in inflammation and cell cycle controlaccounts for these observations.186–188

G. Therapeutic Potential of FXR LigandsStudies in humans with bile acid resins such as cholestyramine that absorb

bile acids indicate that depletion of bile acids decreases cholesterol levels.189

The activity of bile acid resins most likely results from the upregulation ofCYP7A1 and increased catabolism of cholesterol to restore the bile acids thathave been removed (Fig. 5). While these resins are an effective means to lowercholesterol compliance is generally poor. Bile acid resin powders must bemixed with water or fruit juice and taken once or twice daily with meals. Tabletsmust be taken with large amounts of fluids to avoid gastrointestinal symptoms.Resin therapy may also produce a variety of side effects including constipation,bloating, nausea, and gas. The observation that FXR controls the bile acid‐dependent repression of CYP7A1 suggested to many that an FXR antagonistwould block bile acid‐dependent repression of CYP7A1 and produce thebenefits of a bile acid resin without unwanted side effects.132 Guggulsterone,the active ingredient of a naturally occurring medicinal cholesterol loweringagent isolated from the guggul tree, has been reported to be an FXR antago-nist.190 The putative cholesterol‐lowering activity of guggulsterone is consistentwith the predicted activity of an FXR antagonist. Guggulsterone, however,appears to have multiple biological targets and it is not clear if any of thephysiological effects of this compound are derived from inhibiting FXR.190–192

In contrast, all the activities associated with FXR agonists including loweringlipid levels, decreasing atherosclerosis, improving insulin sensitivity, and hepa-toprotection appear to be beneficial. We would suggest that the apparentdisconnect between the activities of bile acid resins (upregulate CYP7A1,lower cholesterol) and FXR agonists (downregulate CYP7A1, lower cholester-ol) lies in the site of the action of these two agents. Bile acid resins function inthe intestine and remain there for extended periods. Depleting bile acids in theintestine will decrease the expression of FGF19, relieve the feedback inhibitionon CYP7A1, and promote the breakdown of cholesterol (Fig. 5). In contrast,the synthetic agonists tested are relatively rapidly absorbed into the bloodstream and accumulate in the liver. In the liver, activation of FXR will induceSHP and strongly inhibit expression of CYP8B1 shifting bile acid compositiontoward MCA/CDCA and limiting cholesterol absorption in the intestine(Fig. 5). Activation of FXR in the liver has the added benefits of promotingbile flow, and protecting against tissue damage. Thus, all the preclinical studiessuggest that FXR agonists have the ideal therapeutic profile for the treatmentof human disease. Currently, 6‐ethyl CDCA is in phase II clinical trial for the


treatment of cholestasis and Wyeth has completed a Phase I clinical study of asynthetic FXR agonist (information on ClinicalTrials.gov). The data from thesestudies are not currently available.

V. RORa

The RAR‐related orphan receptor (ROR) family consists of three members(RORa, NR1F1; RORb, NR1F2; and RORg, NR1F3) that bind to DNA asmonomers to regulate transcription.193–195 ROR response elements (ROREs)consist of a six basepair A/T rich region immediately preceding a half siteAGGTCA motif. RORs are constitutively active transcription factors whichrecruit coactivators in the absence of exogenous ligand.196–199 Nevertheless,crystal structures suggest that these orphan receptors can bind and be regu-lated by ligands. All‐trans retinoic acid was crystallized with LBD of RORb andcan inhibit RORb transcriptional activity in cells, suggesting a possible role ofRORb in regulating retinoid action.200 Interestingly, a structure of RORaobtained using baculovirus‐expressed protein unexpectedly identified choles-terol as a ligand for RORa.201 This suggests that cholesterol or derivatives ofcholesterol might function as physiological ligands for RORa. Based on thisstructure, point mutations of RORa that prevent cholesterol binding weregenerated and the ability of these mutants to activate a reporter gene with anRORE was impaired.201 Further confirmation of cholesterol as a RORa ligandwas provided by decreased RORa activity in cells treated with lovastatinor hydroxypropyl‐B‐cyclodextrin to lower intracellular cholesterol. Underthese cholesterol‐depleted conditions, adding back cholesterol restores RORaactivity.201 Although it cannot be ruled out that the effect of cholesterol isindirect, these results demonstrate that RORa activity can be modulated bycholesterol status in cells. Thus, RORa may act as a lipid sensor of theintracellular cholesterol levels and play an important role in the regulation ofcholesterol homeostasis.

A. Regulation of Lipid Metabolism by RORsRORa is highly expressed not only in blood, brain, skin, intestine but also in

metabolically important tissues such as liver, muscle, and adipose tissue.202 Thephysiological function of RORa in vivo has been revealed using staggerer mice(RORasg/sg). These mice contain a 122‐bp deletion in the coding sequence ofRORa that prevents translation of a functional LBD.203,204 The most obviousdefect in RORasg/sg mice is their balance deficit due to massive neurodegen-eration in the cerebellum, caused by a developmental defect in Purkinje cells.


This cerebellar atrophic phenotype was also found inRORa knockout (RORa�/�)mice, providing validation that the RORasg/sg defects are due to the functionaldisruption of RORa gene.205

The role of RORa in lipid homeostasis and atherosclerosis/obesity wasuncovered by the observation of metabolic abnormalities in RORasg/sg mice.RORasg/sg mice are smaller than wild type despite hyperphagia and have lowertotal and HDL cholesterol levels. The decrease in HDL is thought to arisefrom reduced plasma ApoA‐I and ApoA‐II, both major apolipoproteins ofHDL, in these mice and from decreased expression of ApoA‐I in the intestine,previously shown to be a direct target gene for RORa.206 The reverse choles-terol transporters ABCA1 and ABCG1 genes are also downregulated in theliver from RORasg/sg mice.207 Not surprisingly given the low levels of HDL,when fed with an atherogenic diet RORasg/sg mice are more susceptible todevelop atherosclerosis compared to wild‐type mice.208

Plasma triglycerides levels from RORasg/sg mice are reduced and this iscorrelated with decreased hepatic and intestinal expression of ApoC‐III, acomponent of HDL and VLDL that regulates triglyceride levels and is alsoa RORa target gene.209 Interestingly, a recent genome‐wide association studyindicated that individuals who are heterozygous for a null mutation in ApoC‐IIIhave low plasma triglycerides, a favorable cholesterol profile and decreased riskfor cardiovascular disease.210 Consistent with the lowering of plasma triglycer-ides, liver triglycerides are also found to be decreased in RORasg/sg mice.207

Examination of genes involved in lipogenesis in the liver show that SREBP‐1cand FAS expression are reduced with little change in LXR expression, suggest-ing factors other than LXR may be involved in regulating expression of thesegenes. Using transfection and chromatin immunoprecipitation assays, RORawas shown to bind to the SREBP‐1c promoter and modulate its activity. Thus,the decreased triglyceride levels detected in staggerer mice may be a directresult of RORa deficiency.

Perhaps related to the defect in triglyceride metabolism, RORasg/sg

mice have reduced adiposity associated with decreased fat pad mass andadipocyte size207 and are resistant to diet‐induced weight obesity. Comparedto wild‐type mice, there is a lack of fat accumulation in the gonadal and inguinalwhite adipose and interscapular brown adipose of RORasg/sg mice on highfat diet.207 These results further demonstrate the importance of RORa inlipogenesis in the adipose tissue and liver.

In muscle, overexpression of a dominant negative RORa that lacks theentire LBD and part of the hinge region results in a reduced expression ofSREBP‐1c and other lipogenic genes (FAS, SCD‐1, SCD‐2). Similarly, genesinvolved in lipid and cholesterol efflux and homeostasis (ABCA1, apoE, CAV3)are repressed in these cells versus the wild‐type cells. Other genes involved inlipid absorption (CD36, FABP3), lipid catabolism (LPL, CPT1, ACS4, adipoR1,


adipoR2, PGC‐1), lipid storage (ADRP) are also downregulated.211 Collectively,these in vivo and in vitro studies demonstrate that RORa is an importantmodulator of lipid homeostasis in the intestine, liver, adipose tissue, andmuscle.

The involvement of the other twoROR familymembers, RORb andRORg, inlipid metabolism is less clear. RORb expression is restricted mostly to brain andretina and expressed poorly in other tissues such as liver, muscle, and adiposetissue.194,202,212 RORg is highly expressed in muscle and its function in lipid andglucose metabolism has been examined using the in vitro muscle cell model.In these studies, although expression of a dominant negativeRORg that lacksAF‐2domain significantly represses RORg‐dependent gene expression, genes involvedin lipid metabolism are not affected.213 Similarly, RORg‐deficient mice havenormal triglycerides and cholesterol levels.214 Interestingly, serum glucose levelsare significantly lower in RORg‐deficient mice but normal in staggerer mice,suggesting that RORs have different roles in lipid and glucose homeostasis.214

Rev‐erba is an orphan nuclear receptor which can bind ROREs and act as adominant‐negative repressor of RORs by competing for the sameDNA bindingsites.215 Consistent with the idea that RORs and Rev‐erba compete for bindingsites is the observation that Rev‐erba can repress ApoA‐I and ApoC‐III geneexpression in HepG2 cells and primary hepatocytes.216–218 In Rev‐erba‐deficient mice, serum triglycerides are elevated and this is associated with theincreases in serum apoC‐III concentration and liver apoC‐III RNA levels.217

Since RORs and Rev‐erbs are coexpressed in many tissues, the effects of thesereceptors on lipid metabolism are likely to be determined by the ratio of theirexpression levels, which can be altered by diet or circadian rhythm (see below).

B. Role of RORs in Circadian Rhythm Control andLinks to MetabolismThere is increasing evidence suggesting that regulation of the biological

clock is linked to energy metabolism. Mutation of the CLOCK (Circadianlocomotor output cycles kaput) gene, a key transcription factor of the molecularcircadian clock, results in a phenotype of metabolic disease in mice.219 Homo-zygous Clock mutant mice lose the diurnal rhythm in food intake and theirmetabolic rates are decreased. They gain more weight than wild‐type mice andhave higher triglycerides, cholesterol, and glucose in the serum and accumula-tion of fat in the liver, all hallmarks ofmetabolic syndrome. Conversely, a high fatdiet which induces the same metabolic phenotypes also alters the cyclic expres-sion of genes important for maintaining circadian rhythm,202 emphasizing theinterrelationship of metabolic homeostasis and molecular circadian clock.

Genomic profiling of suprachiasmatic nuclei (SCN) and liver has identifiedan important role of the RORE in gene expression during circadian cycle. Thisimplies that RORs and Rev‐erba may be important transcriptional circadian


regulators.220 Indeed, RORs and Rev‐erba display circadian expression in theSCN and peripheral tissues important for metabolism (liver, muscle, WAT,BAT).202,220 In these tissues, Rev‐erba and Rev‐erbb show similar circadianpattern with highest level at CT (circadian time) 4–8 and lowest at CT16–20.Expression of different ROR isoforms is more tissue‐specific. In the SCN,RORg is not detected and RORb expression is high but shows little oscillationwhereas RORa expression is cyclic with highest level at CT4–12. In the WAT,RORa expression peaks at CT12, right after the peaks of Rev‐erbs expression.In the liver and BAT, RORg expression reaches its maximal level at CT16 andminimal level at CT4, showing an antiphase pattern to Rev‐erbs expression.Interestingly, expression of Bmal1 (Brain and muscle ARNT like protein 1),another important gene involved in regulating circadian clock,221 generallyreaches the lowest level and highest level in both SCN and metabolicallyimportant tissues (liver, muscle, fat) at the peak and trough of Rev‐erbsexpression, respectively (Fig. 6).202,220 Bmal1 gene contains ROREs in itspromoter region and is likely be regulated by RORs and Rev‐erbs. Indeed,Bmal1 has been shown to be a target gene of RORa and RORg and itstranscription is activated by RORs and repressed by Rev‐erbs through compet-ing for the same ROREs (Fig. 6).222–224

The importance of Rev‐erba and RORa in Bmal1 transcription and circa-dian rhythm is further demonstrated in mice deficient in these orphan nuclearreceptors. In wild‐type mice the amplitudes of Bmal1 RNA oscillations in the

0

Rel

ativ

e R

NA

leve

ls

4

ROREs

RORs

Bmal1

Bmal1

Rev-erbs

Rev-erbs

8 12 16 24 CT20

FIG. 6. Schematic representation of circadian Bmal1 expression and its regulation by RORsand Rev‐erbs. RORs and Rev‐erbs bind to the ROREs in the promoter of Bmal1 gene and activateor repress its transcription depending on the relative expression levels of these two classes oforphan nuclear receptors. Both Rev‐erba and Rev‐erbb cycle in an antiphase to Bmal1 in all tissues(SCN, liver, muscle, adipose) whereas three ROR isoforms exhibit different circadian patterns indifferent tissues. Decrease of Rev‐erbs expression in cells may allow the RORs to function as anactivator which is inhibited when Rev‐erbs levels are high.


liver are at least 20‐fold with lowest point at CT8. This cyclic change in Bmal1expression is reduced to less than 2‐fold in Rev‐erba�/� mice.225 Similarly, thedramatic decrease of Bmal1 expression in the SCN at CT4 compared to that atCT16 is also lost in these mice. The Rev‐erba�/� mice have shortened periodlengths of their locomotor activity rhythm in constant darkness or light, and thedistribution of period lengths is much more variable than in wild‐type mice.Interestingly, the more variable and shortened period of locomotor activityrhythm is also seen in the RORasg/sg mice.222,223 In the SCN of the RORasg/sg

mice, the peak Bmal1 expression at CT15–18 is much reduced compared towild‐type mice. These studies suggest that RORa and Rev‐erba are requiredfor maintaining normal cyclic Bmal1 expression and circadian clock function.

The RORasg/sg and Rev‐erba�/� mice, as discussed earlier, have abnormaltriglycerides levels. Plasma triglycerides in mice show circadian rhythm withhighest level at CT4 and lowest level at CT16.226 This cyclic change in trigly-cerides is lost in Bmal1�/� mice, with similar levels at CT4 and CT16. It ispossible that Bmal1 expression, regulated by RORs and Rev‐erbs, may berequired for lipid homeostasis. Mouse embryonic fibroblasts (MEFs) isolatedfrom Bmal1�/� mice and Bmal1‐knockdown 3T3‐L1 cells are resistant toadipocyte differentiation and accumulate less lipids.227 This is correlated witha significantly reduced induction of adipocyte‐related genes such as PPARg andaP2. Reexpression of Bmal1 in Bmal1�/� MEFs can restore the adipocyte geneexpression and lipid accumulation. In mature 3T3‐L1 adipocytes, overexpres-sion of Bmal1 increases lipid synthesis activity which is associated with anincrease in the RNA levels of genes involved in lipid metabolism such asSREBP‐1a, FAS, and aP2. Interestingly, the RORasg/sg mice have reducedadiposity and decreased expression of lipogenic genes.207 The similarity ofthese lipogenic deficiency phenotypes in Bmal1�/� and ROR�/� cells providesanother link between circadian rhythm and lipid metabolism.

C. RORs and InflammationAs we have observed with the other metabolic sensing nuclear receptors,

RORa also appears to play an important role in regulating inflammation. In theimmune system, RORa is expressed in both lymphoid and myeloid cells withhighest expression in macrophages.228 Peritoneal macrophages isolated from theRORasg/sg mice express higher RNA levels of IL‐1b, TNFa, and IL‐1a than wild‐type mice upon LPS stimulation.229 In Rora�/� mice, bone marrow derivedmacrophages also produce more IL‐6 and TNFa than those obtained fromwild‐type mice.228 Since inflammation is also a key element in atherosclerosis,the activity of RORawas examined in primary human aortic smoothmuscle cells.RORa is expressed in these cells and overexpression of RORa inhibits TNFa‐induced IL‐6, IL‐8, and COX‐2 expression.230 In addition, RORa upregulates


gene transcription of IkBa, the major inhibitory protein of the NF‐kB signalingpathway, through a RORE in the IkBa promoter in these primary muscle cells.The role for RORa in regulating IkBa was confirmed in vivo by the significantlylower basal levels of IkBamRNA in aortas from theRORasg/sgmice than thewild‐type mice. These studies suggest that RORa is anti‐inflammatory and may have aprotective role during inflammation and metabolic disease.

D. Therapeutic Potential of RORa LigandsStudies in vivo and in vitro have demonstrated the importance of RORa in

regulating expression of genes that control cholesterol and triglyceride levels aswell as circadian rhythm. Recent studies linking the circadian rhythm to energymetabolism and demonstrating the essential role of ROREs in circadian geneexpression suggest that RORs and their dominant negative regulators Rev‐erbsare likely to be important players in integrating the circadian clock with lipidmetabolism. Identification of cholesterol and ATRA as RORa and RORbligands suggests that it may be possible to develop small molecules that canbind RORs and modulate their activities. Based on the atherosclerotic andinflammatory phenotypes of RORasg/sg mice, a RORa agonist will likely to bebeneficial in improving cardiovascular disease. Nevertheless, an RORa agonistmay also potentially increase lipid synthesis in the adipose and liver. As wehave observed with the other receptors discussed in the review, the challengefor drug discovery targeting RORa will be to identify small molecules thatfunction in a tissue‐selective manner in order to maximize the therapeuticbenefit relative to unwanted side effects.

VI. ERRa

Estrogen receptor related receptora (ERRa; NR1B1) is one of the threemembers of the ERR family (ERRa, ERRb, NR1B2; ERRg, NR1B3) thatbind to the ERR responses element (ERRE) composed of a nuclear receptorhalf‐site preceded by three nucleotides with the consensus sequenceTNAAGGTCA.231 ERRa is most related to estrogen receptor a (ERa), sharinga high degree of homology within their DBD (68% identity). Not surprisingly,ERRa also bind estrogen receptor response elements (ERE) consisting of aninverted repeat of the core AGGTCA separated by three nucleotide.232 Indeed,several ERE‐containing gene promoters can be regulated by both ERa andERRa,232–234 suggesting potential cross talk between the signaling pathwaysmediated by these two receptors.

ERRa is constitutively active, assuming a LBD conformation that resem-bles that of agonist‐bound nuclear receptors in the absence of ligand.235,236

A crystal structure of ERRa complexed with an LXXLL peptide from PGC‐1a


indicates that the LBD of ERRa is almost completely filled with hydrophobicside chains, resulting in a small ligand binding pocket which can only beoccupied by compounds with no more than the equivalent of four or fivecarbon atoms. Based on the sequence similarity between ERRa and ERa,a series of estrogen‐like compounds were screened for their ability to modulateERRa transcriptional activity. The phytoestrogens geneistein, daidzein, andbiochanin as well as 6,30,40‐trihydroxyflavone were identified as agonists ofERRa by a virtual ligand screening using the agonist‐bound ERa as thetemplate.237 The activity of these phytoestrogens on ERRa was confirmed incell transfection assays but they also activate ERs and other ERRs. Using ayeast one‐hybrid assay with an ERRa‐binding DNA region from the humanaromatase gene, two organocholorine (pesticides toxaphene and chlordane)were identified to interact with ERRa.238 These compounds exhibit weakagonist activity on ERa but inhibit transcriptional activity of ERRa in mamma-lian cells. Diethylstilbesterol (DES), a potent ER agonist, was also identified asan antagonist of ERRa with an IC50 at �1 mM using an in vitro coactivatorinteraction assay.239 In cells, DES disrupts the ERRa‐coactivator interactionand inhibits the constitutive transcriptional activity of ERRa. The first syntheticERRa ligand XCT790, and inverse agonist, was derived from high throughputscreening using an in vitro coactivator interaction assay.240,241 This compoundis a potent inhibitor of ERRa transcriptional activity in cells (IC50: 300–500nM) with no significant activity on related nuclear receptors such as ERRg andERa at concentrations below 10 mM, providing a pharmacological tool forunderstanding the functions of ERRa. Recently, another ERRa inverse agonistwas derived from high throughput screening and crystallized with ERRaLBD.242 This compound inhibits the interaction of ERRa and PGC‐1a peptidein a biochemical assay with an IC50 of 190 nM. Binding of this antagonistpositions Helix 12 in the coactivator binding groove where the PGC‐1a peptideis located in the apo‐ERRa crystal structure,236 providing an explanation forthe molecular mechanism of an ERRa inverse agonist. The identification ofnumerous ERRa inverse antagonists and few agonists suggest that the smallligand binding pocket is likely to accommodate only tightly packed moleculesand most compounds that bind will result in a disruption of the ERRa tran-scriptional activity.

A. ERRa and the PGC‐1a PathwayERRa is ubiquitously expressed with higher levels in the tissues (muscle,

kidney, heart, and BAT) that have high metabolic needs.243,244 This expressionpattern of ERRa is very similar to that of PGC‐1a (which has been shown toinduce ERRa gene expression through the ERRE in the promoter region andalso function as an ERRa coactivator).245,246 Physiological stimuli such as


starvation, exposure to cold temperatures, and exercise increase both PGC‐1aand ERRa gene expression,244,246,247 suggesting that these two proteins maymediate similar cellular functions.

PGC‐1a plays an important role in regulating energy metabolism andmitochondrial biogenesis in muscle and BAT.248,249 In the course of identifyingtranscription factors and their cis‐regulatory elements that are important inmediating the activities of PGC‐1a, ERR binding sites (ERREs) were found tobe one of the key motifs enriched in the genes upregulated by PGC‐1a inmuscle cells.250 This suggests that an important function of PGC‐1a in muscleis to modulate transcriptional activity of ERRs. The ERRE motif is highlyenriched in the promoters of genes involved in oxidative phosphorylationwhich are also targeted by PGC‐1a.249 The role of ERRa in the regulation ofPGC‐1a‐mediated mitochondrial functions was demonstrated by using theERRa‐specific antagonist XCT790.250 In C2C12 myotube cells, PGC‐1aincreases expression of ERRa and genes involved in oxidative phosphorylationbut XCT790 diminishes this induction. XCT790 also significantly reduces thePGC‐1a‐increased total mitochondrial respiration. The requirement of ERRain the PGC‐1a mediated mitochondrial biogenesis is demonstrated in anosteosarcoma cell line SAOS2.251 Overexpression of PGC‐1a in these cellsinduces genes encoding mitochondrial proteins such as mitochondrial DNAreplication and transcription (mtTFA), cytochrome c, somatic (Cyt c), and ATPsynthase b (ATPsynb), and mitochondrial DNA contents are also increased.Inhibition of ERRa expression using an siRNA approach results in the decreasein expression of these mitochondrial proteins and functions mediated byPGC‐1a. In these studies, ERRa is also shown to bind to the ERREs in thepromoters of ATPsynb and Cyt c genes.

PGC‐1a also plays a role in regulating glucose and fatty acid metabolism inan ERRa‐dependent manner. Overexpression of PGC‐1 in C2C12 myotubecells induces gene expression of pyruvate dehydrogesnase kinase 4 (PDK4),which contains an ERRE in the promoter region.252 PGC‐1a‐mediated activa-tion of the PDK4 promoter requires ERRa since this activity is not seen whenusing MEFs from Erra�/� mice compared to those from the wild‐type mice.Stimulation of PDK expression suggests that glucose oxidation is inhibited andenergy utilization is switched from glucose to fatty acids. Indeed, PGC‐1aexpression in C2C12 myotube cells decreases the glucose oxidation rate andis accompanied by an increase in the expression of median‐chain acylcoenymeA dehydrogenase (MCAD), an enzyme that mediates the first step in themitochondrial b‐oxidation of fatty acids. MCAD is a target gene of ERRa andcoexpressed with ERRa and PGC‐1 in tissues that preferentially utilize fattyacids as energy substrates.243 The PGC‐1‐induced MCAD expression can beinhibited using the ERRa‐specific inhibitor XCT790,250 further demonstratingthe requirement of ERRa in PGC‐1‐mediated fatty acid oxidation.


Another metabolic function of PGC‐1a affected by ERRa is its activity ongluconeogenesis in the liver. Overexpression of PGC‐1a in primary hepatocytesincreases expression of gluconeogenic enzymes phosphoenolpyruvate carboxy-kinase (PEPCK) and glucose‐6‐phosphatase (G6Pase) and results in increasedglucose production.253 Injection of an adenovirus expressing PGC‐1a into ratsalso increases blood glucose levels, associated with increases in PEPCK andG6Pase expression. Similarly, PGC‐1a also increases PEPCK gene expressionin HepG2 cells and this is accompanied by an increase in ERRa expression aspredicted.254 However, coexpression of ERRa decreases this PGC‐1a‐mediated induction of PEPCK which contains an ERRE in its promoterregion, suggesting ERRa functions as a transcriptional repressor of PGC‐1ain this context. Indeed, inhibition of ERRa expression using a siRNA increasesPGC‐1a‐induced PEPCK expression in HepG2 cells. The inhibitory activity ofERRa on PGC‐1‐mediated gluconeogenesis is further confirmed by the signif-icantly increased expression of PEPCK and glycerol kinase genes in livers fromthe Erra�/� mice compared to the wild‐type mice. Interestingly, ERRa stillfunctions as an activator of PGC‐1a genes involved in the oxidative phosphory-lation in the liver since the PGC‐1a‐induced mitochondrial gene expression inHepG2 cells is not activated but inhibited by ERRa siRNA. This suggests thatERRa represses gene expression in a promoter‐specific manner and is not ageneral inhibitor in the liver.

B. ERRa and DiabetesPGC‐1a and the genes involved in the oxidative phosphorylation are

decreased in human diabetic muscles, suggesting that muscle mitochondrialdysfunction is linked to diabetes.255 PGC‐1a is also increased in the livers ofdiabetic mouse models, supporting the hypothesis that increased glucoseproduction stimulated by PGC‐1a has a negative impact on insulin sensitivi-ty.253 The ability of PGC‐1a to increase expression of genes involved in bothmitochondrial oxidative phosphorylation in muscle and gluconeogenesis in liversuggest that tissue‐specific modulation of PGC‐1a will be needed to achieve anet beneficial effect for the treatment of diabetes. However, the ability of ERRato activate PGC‐1a‐induced oxidative phosphorylation in muscle and repressPGC‐1a‐mediated gluconeogenesis in liver (as discussed earlier) presents anopportunity to increase mitochondrial function without the detrimental effecton glucose production by targeting ERRa.

C. ERRa in Adipose and IntestineErra�/� mice have a 50–60% reduction in the weight of the inguinal,

epididymal, and peritoneal fat pads with decreased adipocytes size.256 Thesemice are also resistant to high fat diet‐induced obesity. Microarray analysis of


the adipose tissues from ERRa�/� mice show downregulation of the genesinvolved in fatty acid synthesis such as FAS, and SREBP1. Lipogenesis in theErra�/� mice assessed by treating with 3H2O and measuring the amount ofradioactive label incorporated into triacylglycerol show that there is a 30–50%decrease in 3H incorporation into lipids in the inguinal, epidymal, and perirenalfat. These results indicate that ERRa is important for normal lipid metabolismin the white adipose tissue.

In the brown adipose tissue (BAT) of Erra�/� mice, expression of mito-chondrial genes important for oxidative phosphorylation and fatty acid oxida-tion is decreased, demonstrating the importance of ERRa in mediatingmitochondrial energy metabolism in this tissue.257 Mitochondrial density andDNA contents are also decreased in the BAT of Erra�/� mice compared to thewild‐type mice, confirming the in vivo function of ERRa in mitochondrialbiogenesis. The physiological significance of the decreased mitochondrialfunction and mass is shown by the inability of Erra�/� mice to maintain properbody temperature when exposed to cold, suggesting a defect in adaptivethermogenesis.

The lean phenotype of Erra�/� mice arises without decreased food con-sumption or increased energy expenditure.256 One possible explanation for thephenotype of these mice is a defect in intestinal adsorption of dietary nutrientsincluding fat. ERRa is expressed in epithelia cells throughout the small intes-tine244,258 and fat malabsorption is observed in the Erra�/� pups.256 Micro-array analysis of intestinal RNA from Erra�/� mice show that genes involvedin oxidative phosphorylation are downregulated and intestinal b‐oxidationactivity is also reduced, confirming the in vivo role of ERRa in mitochondrialmetabolism in this tissue.258 Additionally, expression of genes involved indietary lipid digestion and absorption such as pancreatic lipase‐related protein 2(PLRP2), fatty acid‐binding protein 1 and 2 (L‐FABP and I‐FABP), andapolipoprotein A‐IV (apoA‐IV) are also downregulated in the intestine ofErra�/� mice.256

D. ERRa and CancerThe observation that ERRs and ERs can bind to the same DNA sequences

suggests that ERRs may influence well characterized estrogen‐dependentpathways. Interestingly, ERRa has been identified as a negative prognosticfactor for breast cancers. ERRa is expressed in many breast cancer cell linesincluding the ER‐positive MCF‐7 and ER‐negative MDA‐MB‐231 cells. Treat-ment of these cell lines with high concentrations of DES, which antagonizesERR activity, inhibits cell proliferation.234 Knockdown of ERRa expression inthe MDA‐MB‐231 cells using siRNA also results in a reduction in cell migra-tion in vitro and tumor growth when implanted as xenografts into athymic


nude mice.259 Recently, the angiogenic vascular endothelial growth factor(VEGF) was identified as a direct target gene for ERRa, providing anotherevidence linking ERRa to tumor progression and metastasis.260 These resultssuggest that pharmacological manipulation of ERRa activity may be of thera-peutic use in treating breast cancer patients.

E. Therapeutic Potential of ERRa LigandsAlthough Erra�/� mice exhibit a relatively mild metabolic phenotype this

could be due to the upregulation of compensatory pathways. Indeed, expressionof PGC‐1a is increased in the heart, liver, andmuscle ofErra�/�mice andERRgis also upregulated in the heart of these mice.254,261 Several studies indicatemitochondrial oxidative phosphorylation is reduced in patients with type IIdiabetes255,262 and ability of ERRa to induce mitochondrial biogenesis andoxidative phosphorylation in conjunction with PGC‐1a suggests the straightforward hypothesis that an ERRa agonist would be useful for the treatment ofthis disease (Fig. 7). Nevertheless, this simple hypothesis is confounded by therecent observation that over expression of PGC‐1a in muscle actually promotesinsulin resistance.263 Additionally, to our knowledge none of the compoundsreported to have ERR agonist activity in vitro and in cell culture systems havebeen shown to act as ERR agonists in animals. Hydrophobic amino acidresidues in the LBD of ERRa appear to function as an intramolecular agonistfavoring a transcriptionally active conformation and we feel it is unlikely thatsynthetic agonists with the efficacy to significantly influence gene expression inanimals will be identified. On the contrary, ERRa antagonists that disrupt theactive confirmation have been identified240–242,250 and we anticipate that thefurther development of such inhibitors with sufficient systemic exposure willhelp elucidate the in vivo function of ERRa. Clearly, such inhibitors may havetherapeutic value for the treatment of cancer and one wonders about themetabolic impact of such ligands. Will inhibiting ERRa function to decrease

Gluconeogenesis (e.g., PEPCK)

Fatty acid oxidation (e.g., MCAD)Glucose oxidation (e.g., PDK4)Lipid transport (e.g., ApoA-IV)

Oxidative phosphorylation (e.g., Cyt c)

Tumor growth (e.g., pS2)

Angiogenesis (e.g., VEGF)ERRa

FIG. 7. ERRa activity and its target genes. ERRa binds to the ERREs in the promoters of itstarget genes and activates or represses their expression. The mitochondrial functions and otheractivities of ERRa have been characterized using a combination of the Erra�/� mice, siRNA, andERRa‐specific antagonists.


lipid absorption perhaps having a beneficial effect on plasma lipids or willdecreases in oxidative phosphorylation speed the progression of diabetes(Fig. 7)?

VII. Summary

Over the last 15 years a growing number of nuclear hormone receptorshave been demonstrated to play important roles in the control of metabolism atthe level of gene expression. Nature has seized on the ability of nuclearreceptors to function as ligand‐controlled transcription factors and exploitedthis ability to evolve a family of metabolic sensors that coordinate metabolismin response to changes in the levels of important end‐products and intermedi-ates. Recent studies have also defined an intimate link between the regulationof metabolism and the control of inflammation. Excitingly, many of the recep-tors highlighted in this review appear to function at this interface. Although thebiological necessity for linking metabolism with inflammation is not yet clear,the best evidence suggests that control of energy utilization plays an importantrole in determining inflammatory status. Since many of the pathways regulatedby the PPARs, LXRs, FXRs, RORs, and ERRs are deranged in states ofmetabolic disease these receptors have become important drug targets fordevelopment of novel drugs for human disease. Indeed, preclinical and clinicalstudies have uncovered many beneficial activities for small molecules thatregulate these receptors. Nevertheless, in many cases there also appear to beunwanted activities of many of these small molecules that can or may limit theirtherapeutic use. Future studies that better define the tissue‐specific activitiesof the metabolic nuclear receptors and the trans‐acting factors that interactwith receptors to impart tissue‐selective gene expression patterns will pave theway toward the identification of effective drugs for metabolic disease.

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regulation of metabolism by nuclear hormone …...this review will be on the function of these...

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