26 Dec 2002 18:33 AR AR177-PH65-11.tex AR177-PH65-11.sgm LaTeX2e(2002/01/18) P1: IBC 10.1146/annurev.physiol.65.092101.142528 Annu. Rev. Physiol. 2003. 65:261–311 doi: 10.1146/annurev.physiol.65.092101.142528 c 2003 by Annual Reviews. All rights reserved Copyright ° First published online as a Review in Advance on October 18, 2002 NUCLEAR RECEPTORS AND THE CONTROL OF METABOLISM Gordon A. Francis1, Elisabeth Fayard2, Frédéric Picard2, and Johan Auwerx2 1 CIHR Group on Molecular and Cell Biology of Lipids and Departments of Medicine and Biochemistry, 328 HMRC, University of Alberta, Edmonton, Alberta, Canada T6G 2S2; 2 Institut de Génétique et Biologie Moléculaire et Cellulaire (IGBMC), CNRS/INSERM/ULP, 67404 Illkirch, France; e-mail: auwerx@titus.u-strasbg.fr Key Words atherosclerosis, adipose tissue, cholesterol, gene expression, transcription ■ Abstract The metabolic nuclear receptors act as metabolic and toxicological sensors, enabling the organism to quickly adapt to environmental changes by inducing the appropriate metabolic genes and pathways. Ligands for these metabolic receptors are compounds from dietary origin, intermediates in metabolic pathways, drugs, or other environmental factors that, unlike classical nuclear receptor ligands, are present in high concentrations. Metabolic receptors are master regulators integrating the homeostatic control of (a) energy and glucose metabolism through peroxisome proliferator-activated receptor gamma (PPARγ ); (b) fatty acid, triglyceride, and lipoprotein metabolism via PPARα, β/δ, and γ ; (c) reverse cholesterol transport and cholesterol absorption through the liver X receptors (LXRs) and liver receptor homolog-1 (LRH-1); (d ) bile acid metabolism through the farnesol X receptor (FXR), LXRs, LRH-1; and (e) the defense against xeno- and endobiotics by the pregnane X receptor/steroid and xenobiotic receptor (PXR/SXR). The transcriptional control of these metabolic circuits requires coordination between these metabolic receptors and other transcription factors and coregulators. Altered signaling by this subset of receptors, either through chronic ligand excess or genetic factors, may cause an imbalance in these homeostatic circuits and contribute to the pathogenesis of common metabolic diseases such as obesity, insulin resistance and type 2 diabetes, hyperlipidemia and atherosclerosis, and gallbladder disease. Further studies should exploit the fact that many of these nuclear receptors are designed to respond to small molecules and turn them into therapeutic targets for the treatment of these disorders. 0066-4278/03/0315-0261$14.00 261 26 Dec 2002 18:33 262 AR AR177-PH65-11.tex AR177-PH65-11.sgm LaTeX2e(2002/01/18) P1: IBC FRANCIS ET AL. NUCLEAR RECEPTORS General Concepts Nuclear receptors are one of the largest groups of transcription factors, with 49 distinct members presently identified in the human genome (1, 2). The activity of many nuclear receptors is controlled by the binding of small, lipophilic ligands that include hormones, metabolites such as fatty acids, bile acids, oxysterols, and xenoand endobiotics. Several of these receptors were characterized before their ligands were identified, and because of this were designated orphan receptors. As their natural and synthetic ligands have become known, many of these orphans have now been adopted (3). Recognition of the importance of nuclear receptors as master regulators of genes involved in metabolic control has resulted in the intensive search for novel ligands for these receptors that might be used in preventive and therapeutic strategies to combat common diseases such as atherosclerosis, diabetes, and obesity.1 Structure and Dimerization Nuclear receptors have a modular structure characterized by a ligand-independent AF-1 transactivation domain in the N-terminal region, a highly conserved DNAbinding domain composed of two zinc fingers recognizing specific DNA sequences, and a ligand-binding and dimerization domain that contains a liganddependent AF-2 transactivation domain in its C-terminal portion (4, 5). Most nuclear receptors are active as dimers, either homodimers or heterodimers with the retinoid X receptor (RXR, NR2B1), although a subset of them can bind and stimulate transcription as monomers. The nuclear receptors controlling fat, glucose, cholesterol, bile acid, and xenobiotic metabolism, including the peroxisome proliferator-activated receptors (PPARs), liver X receptors (LXRs), farnesol X receptor (FXR), and the pregnane X receptor (PXR)/steroid and xenobiotic receptor (SXR), all form obligate heterodimers with RXR, and ligand binding to one or the 1 Abbreviations: ABCA1, ATP-binding cassette transporter AI; apoA-I, apolipoprotein A-I; apoC-II, apolipoprotein C-II; apoE, apolipoprotein E; BSEP, bile salt export pump; CETP, cholesterol ester transfer protein; CYP3A, cytochrome P4503A; CYP27, sterol-27 hydroxylase; CYP7A1, cholesterol 7α-hydroxylase; CYP8B1, sterol 12α-hydroxylase; FXR, farnesol X receptor; HDL, high-density lipoprotein; HNF4-α, hepatic nuclear factor 4α; I-BAT, ileal bile acid transporter; I-BABP, ileal bile acid–binding protein; LCA, lithocholic acid; LCAT, lecithin:cholesterol acyltransferase; LDL, low-density lipoprotein; LPL, lipoprotein lipase; LRH-1, liver receptor homolog-1; LXR, liver X receptor; NTCP, sodium taurocholate cotransporting polypeptide; OATP, organic anion transporting polypeptide; PLTP, phospholipid transfer protein; PPAR, peroxisome proliferator-activated receptor; RCT, reverse cholesterol transport; RE, response element; RXR, retinoid X receptor; SHP, small heterodimer partner; SREBP, sterol regulatory-element-binding protein; PXR/SXR, rodent pregnane X receptor/human steroid and xenobiotic receptor; TNFα, tumor-necrosis-factoralpha; VLDL, very-low-density lipoprotein; PCN, pregnenolone-16-alpha-carbonitrile. 26 Dec 2002 18:33 AR AR177-PH65-11.tex AR177-PH65-11.sgm LaTeX2e(2002/01/18) NUCLEAR RECEPTORS IN METABOLISM P1: IBC 263 other partner stabilizes the heterodimer association (6). Unlike several other RXR heterodimer partners (e.g., the retinoic acid receptor RAR and vitamin D receptor VDR), most of these RXR partners may be activated permissively by binding RXR ligands alone (7–10) but are synergistically activated by simultaneous binding of ligands for both dimer partners (5, 6). Corepressors and Coactivators In general, unactivated nuclear receptors form a complex with corepressors [e.g., RIP 140 (11), NcoR (12) and SMRT (13)], which inhibit their transcriptional activity, often through the recruitment of histone deacetylases (14). Activation of the receptor by ligand binding and/or phosphorylation induces a conformational change, resulting in the dissociation of the corepressors and the recruitment of coactivator complexes that facilitate target gene transcription. Coregulator recruitment is therefore an integral part of nuclear receptor signaling pathways (15). The activities of these coregulators range from chromatin remodeling and modifications of core histones (acetylation, phosphorylation, and methylation status) that allow transcription to occur, to recruitment of the basic transcriptional machinery (14). Coactivators that have been shown to interact with nuclear receptors include the p160 family (SRC-1/TIF2/GRIP-1/ACTR) (16–18), p/CIP (19), p300/CBP (20, 21), PGC-1 (22), PRIP (23), PGC-2 (24), and ARA70 (25). The specific coregulators recruited to the nuclear receptors depend on the conformational changes induced by the particular ligand bound and also the promoter and cellular context. Specific receptor/coregulator complexes are responsible for fine-tuning the biological response to the ligand-receptor interaction and underlie the variability of gene responses to different ligands and metabolic environments. METABOLIC NUCLEAR RECEPTORS: TISSUE DISTRIBUTION AND KNOWN LIGANDS PPARs One of the first drugs developed for lipid lowering, clofibrate, was noted to induce proliferation of the cell organelles peroxisomes in rodents (26, 27). The receptor activated by clofibrate and several other peroxisome proliferators that transactivate numerous genes controlling fatty acid oxidation was subsequently cloned and given the name peroxisome proliferator-activated receptor alpha (PPARα) (28). PPARβ/δ and PPARγ were subsequently identified as structural homologs of PPARα that control expression of other metabolic genes but do not induce peroxisome proliferation (29–31). Although PPARα agonists induce fatty acid oxidation in humans, they also do not induce proliferation of peroxisomes as in rodents (32), but the name PPAR has stuck. Initially thought to be a unique isoform, mammalian PPARδ appears to be homologous with PPARβ from other species, and they are currently believed to be the same receptor (32). Throughout this review, this isoform 26 Dec 2002 18:33 264 AR AR177-PH65-11.tex AR177-PH65-11.sgm LaTeX2e(2002/01/18) P1: IBC FRANCIS ET AL. is referred to as PPARβ/δ. The PPAR subfamily of nuclear receptors all bind as heterodimers with RXR to peroxisome proliferator response elements (PPRE) in the target gene, preferentially consisting of a direct repeat of the hormone receptor response element half site spaced by one nucleotide (DR-1) (6). PPARα (NR1C1) has been cloned and characterized in several species including humans (33). PPARα is expressed in metabolically active tissues including liver, kidney, heart, skeletal muscle, and brown fat (34, 35). It is also present in monocytes and vascular endothelial and smooth muscle cells (36–38). Fatty acids are the main natural ligands of PPARα, which then activates the genes for fatty acid catabolism (39–42). High-fat diets, particularly those rich in very long chain fatty acids and polyunsaturated fatty acids (e.g., DHA, EPA), induce fatty acid β-oxidation via PPARα-dependent gene regulation and variable degrees of peroxisomal proliferation in rodents (43). Eicosanoids, derived from arachidonic acid via either the lipoxygenase or cyclooxygenase pathways, also act as PPARα ligands (43). The hypolipidemic amphipathic carboxylic acids or fibrates were the first known synthetic ligands of PPARα. Other synthetic ligands for PPARα include phthalate ester plasticizers, herbicides, food flavors, and leukotriene D4 receptor antagonists (43, 44). Liver PPARα expression follows a diurnal rythmicity and is stimulated under conditions of stress. The changes in PPARα expression in both these situations are directly linked to variations in blood glucocorticoid concentrations, which induce PPARα transcription in a glucocorticoid receptor-dependent fashion (45). This indicates that situations of stress can enhance the impact of nutritional factors on metabolic processes. PPARα activity is also enhanced by protein kinase-A dependent phosphorylation (46). PPARα PPARβ/δ PPARβ/δ (NR1C2) has also been cloned in humans (31) and is expressed ubiquitously, with highest levels found in brain, adipose tissue, and skin (34, 47). Fatty acids and cyclooxygenase 2-derived prostacyclin (PGI2) are natural ligands for PPARβ/δ (41, 48), but PPARβ/δ-selective natural ligands have not yet been identified. Synthetic ligands for PPARβ/δ include bezafibrate (49), furanconjugated linoleic acid metabolite (50), and the recently described more specific high-affinity ligands L-165041 and GW-501516 (51, 52). Although the function of PPARβ/δ is less well known than for the other PPARs, roles for PPARβ/δ have been proposed for embryo implantation (48), skin proliferation and differentiation (53–55), pre-adipocyte proliferation (56), and as a modulator of PPARα and PPARγ activity (57, 58). PPARβ/δ-null mice showed lower fetal and post-natal body weight, diminished gonadal fat stores, and altered epidermal cell proliferation and corpus collosum myelination in one study (53). Impaired placentation and >90% embryonic lethality was seen in two other independent studies (54, 59). Three different PPARγ (NR1C3) mRNAs have been characterized in humans (60, 61). PPARγ 1 and PPARγ 3 transcription gives rise to the PPARγ 1 protein. PPARγ 2 mRNA encodes for an additional 28 amino acids at its N-terminal PPARγ 26 Dec 2002 18:33 AR AR177-PH65-11.tex AR177-PH65-11.sgm LaTeX2e(2002/01/18) NUCLEAR RECEPTORS IN METABOLISM P1: IBC 265 part (62, 63). PPARγ 1 is expressed to a high extent in adipose tissue, large intestine, and hematopoietic cells. Kidney, liver, skeletal and smooth muscles, pancreas, and small intestine express lower amounts of PPARγ 1 (35, 64–66). The PPARγ 2 isoform is restricted to white adipose tissue, where it represents approximately 30% of the PPARγ population. PPARγ 3 has been reported only in the large intestine and macrophages (61). In rodents, adipose tissue PPARγ mRNA and protein levels are reduced after fasting (67, 68) and in streptozotocin-induced diabetes (68), consistent with a stimulatory effect of insulin on PPARγ expression (69). A high-fat diet increases PPARγ expression in rodents (68) and humans (70). Most interestingly, compared with that of the subcutaneous fat depot, the relative expression of PPARγ is increased in visceral fat of obese subjects (71). The natural ligands for PPARγ include fatty acids and fatty acid derivatives (72), such as 15-deoxy-delta12,14-prostaglandin J2 (73), eicosapentaenoic acid (74), 9- and 13-hydroxyoctadecadienoic acids (75). Several chemical classes of synthetic PPARγ ligands have been described. Thiazolidinediones (73, 76) are the best characterized synthetic PPARγ ligands and are currently in use in the clinical management of type 2 diabetes. Other synthetic PPARγ ligands include L-tyrosine-based ligands (77), FMOC-L-leucine (78), and certain non-steroidal anti-inflammatory molecules (79). In addition to modulation by ligand binding, PPARγ activity is also affected by phosphorylation (80, 81). Phosphorylation by MAP kinase, which induces the recruitment of the corepressors SMRT (82), reduces receptor activity in some (80, 81, 83) but not all studies (84). LXRα and β (NR1H3 and NR1H2) Two different LXR genes have been described, LXRα (RLD-1 or NR1H3) and LXRβ (NER, UR, OR-1 or NR1H2). LXRα is expressed to a high extent in liver with lower levels being present in intestine, kidney, spleen, and adipose tissue (35, 85). Although LXRβ is more ubiquitously expressed, less is known about its function. Both LXR isoforms bind preferentially as heterodimers with RXR to DR4 response elements in the genes they activate (85–88). LXRs’ natural ligands are oxysterols (89, 90) and 6α-hydroxy bile acids (91). 24, 25(S)-epoxycholesterol, produced from squalene by a shunt in the classical cholesterol biosynthesis pathway, accumulates in the liver after cholesterol feeding and is the most effective natural LXR ligand. Another potent activator is 22(R)-hydroxycholesterol, which is synthesized in the gonads, adrenals, and placenta. Other molecules that elicit an LXR response are 20(S)-hydroxy-cholesterol and 24(S)-hydroxycholesterol or cerebrosterol, produced to a high extent in developing brain (92), and mevalonate, an intermediate of cholesterol biosynthesis (93). Structure activity studies using oxysterols demonstrated that position-specific monooxidation of the sterol side chain is a requisite for LXR binding and activation (94). Sterols with a 24-oxo group, which act as hydrogen bond acceptors, bind and activate LXR more effectively (94). Furthermore, introduction of an oxygen on the sterol B-ring results in ligands with increased LXRα selectivity (94). Synthetic 26 Dec 2002 18:33 266 AR AR177-PH65-11.tex AR177-PH65-11.sgm LaTeX2e(2002/01/18) P1: IBC FRANCIS ET AL. non-steroidal agonists of LXR have been developed and were instrumental in identifying the induction of SREBP-1c-dependent increases in fatty acid synthesis and stimulation of reverse cholesterol transport by LXR (95). In addition to activation by ligands, LXR activity can also be modulated though phosphorylation by PKA and PKC (96, 97). FXR (NR1H4) Mammalian FXR (98) [RIP-14 in mouse (99)] was initially identified as a homolog of the Drosophila ecdysone receptor (EcR or NR1H1). FXR, whose main role is in bile acid metabolism, heterodimerizes to RXR and binds to DNA sequences consisting of an inverted repeat spaced by one nucleotide (IR-1) (100). FXR is mainly expressed in the liver, gut, kidney, and adrenal cortex. FXR was first shown to be activated by a large variety of endogenous isoprenoids, including farnesol (98), by all-trans-retinoic acid, and by synthetic retinoids such as TTNPB (101). Although these compounds could moderately induce the activity of FXR, none was shown to be direct ligands for FXR. An important breakthrough was the discovery that FXR binds and is activated by several bile acids, including chenodeoxycholic acid, lithocholic acid (LCA), and deoxycholic acid (102–104). Due to this fact, FXR has also earned the name nuclear bile acid receptor (BAR). Bile acids are mostly conjugated to glycine or taurine, a derivative of cysteine. Unlike the non-conjugated bile acids chenodeoxycholic acid, LCA, and deoxycholic acid, their conjugated forms are capable of activating FXR only if the ileal bile acid transporter (I-BAT) is cotransfected in cells, thus enabling their access to the cell (102, 104). This control of ligand entry into the cell restricts receptor activation to cells expressing the transporter protein. PXR (NR1I2) The pregnane X receptor (PXR), also called SXR for steroid and xenobiotic receptor, has been cloned and characterized in human, mouse, rabbit, and rat (8, 105–109). This nuclear receptor exists as two isoforms in the mouse, PXR.1 and PXR.2, that are differentially activated by ligands (8). PXR binds to DNA as a heterodimer with RXR to a DR3 and IR6. It is highly expressed in the liver and at more moderate levels in the intestine, tissues that are important sites for the metabolism of both endogenous and exogenous chemicals. PXR is a promiscuous receptor activated by a wide variety of compounds, including natural and synthetic steroids such as pregnelenone, progesterone, phytoestrogens, dexamethasone, and antiglucocorticoids (8, 105–108, 110), and a number of drugs and plant products such as hyperforin, as well as bile acids including LCA (111–114) and ursodeoxycholic acid (113). Interestingly, high concentrations of synthetic rexinoids activate PXR/RXR in human and rabbit, but not in rat or mouse, through direct binding to PXR (107). There are other marked species differences in the capacity of compounds to activate PXR and induce CYP3A gene expression (105, 108, 114). For instance, 26 Dec 2002 18:33 AR AR177-PH65-11.tex AR177-PH65-11.sgm LaTeX2e(2002/01/18) NUCLEAR RECEPTORS IN METABOLISM P1: IBC 267 the antibiotic rifampicin, which is involved in numerous drug-drug interactions as a consequence of its capacity to induce drug metabolism by CYP3A4, the antidiabetic drug troglitazone, and the hypocholesterolemic drug SR12813 are all efficacious activators of human and rabbit PXR, but have little effect on rat and mouse PXR activity (107). Conversely, pregnenolone-16α-carbonitrile (PCN), a synthetic antiglucocorticoid that was identified for its ability to induce protection from various forms of intoxicants in rodents (115), is a more potent activator of the rat and mouse PXR than the human and rabbit receptor. These speciesspecific differences in PXR activation are based on the limited degree of sequence conservation between mouse, rat, rabbit, and human PXRs, which share only 75– 80% identity in their lipid-binding domain. In transgenic mice expressing human PXR, the pattern of CYP3A inducibility becomes human (116), and they are no longer responsive to mouse-specific PXR ligands. Therefore, these mice constitute valuable in vivo toxicology testing models for the bio-pharmaceutical industry. LRH-1 (NR5A2) The liver receptor homolog 1 or LRH-1, an orphan nuclear receptor, acts as a tissuespecific competence factor and was initially identified by phylogenetic tree analysis of the Ftz-F1 related subgroup of nuclear receptors in Drosophila (117). Homologs of the LRH-1 protein have now been identified in several species (118–121). The human LRH-1 has been reported as pancreas homolog receptor 1 (PHR-1), human FTF (fetoprotein × transcription factor) (122), human B1-binding factor (hB1F) (123), and CYP 7A promoter-binding factor (CPF) (124). Except for FTF, all cDNAs corresponding to human LRH-1 are identical. At least two isoforms have been identified for human LRH-1, suggesting that LRH-1, like other nuclear receptors, is susceptible to alternative splicing to generate isoforms with distinct regulatory functions. In contrast to most of the other receptors discussed in this review, LRH-1 does not dimerize with RXR but binds to DNA as a monomer to an extended nuclear receptor half site (125) in the promoter region of its target genes. These target genes include several critical for bile acid synthesis (124, 126, 127), α-fetoprotein (128), SHP (129), cholesteryl ester transfer protein (130), multidrug resistance protein 3 (131), aromatase (132), and 11-β-hydroxylase (133). LRH-1 expression is mainly confined to liver, exocrine pancreas, and intestine, all derivatives of the gut endoderm (125). LRH-1 has also been found in the ovary (134). LRH-1 is expressed very early in development, with initial expression detected on day 8 in the mouse yolk sac endoderm, and later on during development in the foregut endoderm and the neural crest. During the differentiation of these organs from the foregut, LRH-1 becomes progressively expressed in the epithelial cells of the liver, intestine, and both endocrine and exocrine pancreas. It is only at later embryonic stages (day 17) that LRH-1 starts to exhibit the adult profile when expression is switched off in the endocrine pancreas and in the gut where it becomes restricted to the intestinal crypts of Lieberkuhn 135; J.S. Annicotte & J. Auwerx, unpublished data). No ligand(s) for LRH-1 have yet been identified. 26 Dec 2002 18:33 268 AR AR177-PH65-11.tex AR177-PH65-11.sgm LaTeX2e(2002/01/18) P1: IBC FRANCIS ET AL. SHP (NR0B2) The small heterodimer partner (SHP) is an atypical orphan member of the nuclear receptor superfamily that contains the dimerization and ligand-binding domain found in other members but lacks the conserved DNA-binding domain. The closest relative of SHP in the receptor superfamily is DAX-1, the only other nuclear receptor that lacks the DNA-binding domain. SHP was isolated by yeast twohybrid techniques on the basis of its ability to dimerize with other nuclear receptors (136). It is expressed in the liver, small intestine, spleen, heart, pancreas, adrenal glands, ovary, and testis (136, 137). SHP interacts with a variety of nuclear receptors, including PPARα (138), ER (139), LRH-1 (140–142), LXR (143), and the hepatocyte nuclear factor HNF-4α (144). In most cases, SHP inhibits the activity of the nuclear receptors with which it interacts (136). In addition, SHP can act as a direct transcriptional repressor (145). However, it is puzzling that SHP can enhance the transcriptional activation by both PPARγ (146) and PPARα (147) under particular conditions. Genetic variation in the SHP gene, leading to the loss of SHP activity, is associated with mild obesity (148). Interestingly, SHP mutations seem also to correlate with high birth weight. This phenotype could be related to the effects of SHP on several nuclear receptors associated with metabolic regulation. No ligand has yet been identified for SHP. THE METABOLIC RECEPTORS, METABOLIC SENSORS IN HEALTH AND DISEASE The nuclear receptors discussed above and regrouped under the general name metabolic receptors are primarily involved in the homeostatic control of metabolism and in the defense against toxic compounds. In fact, this subgroup of receptors seems to act as metabolic and toxicological sensors, enabling the organism to adapt quickly to environmental changes by inducing the appropriate metabolic pathways aimed to optimally protect them from overexposure. Ligands for these metabolic receptors are in general products from dietary origin, intermediates in metabolic pathways, drugs, or other environmental factors. Often these compounds are present at high concentration (µM range), which is in sharp contrast to the ligands for the classical nuclear receptors, which are endocrine signaling molecules, generated in minute quantities (nM range). Unfortunately, a westernized lifestyle, characterized by the intake of high-caloric diets, the exposure to drugs and xenobiotics, and a lack of physical exercise, exposes people to chronically higher levels of ligands for these metabolic receptors. Altered signaling by metabolic receptors, triggered by these environmental disruptions, can therefore alter the homeostatic control circuits controlled by these nuclear receptors and contribute to the pathogenesis of many common metabolic diseases, such as obesity, insulin resistance, type 2 diabetes, hyperlipidemia, atherosclerosis, and gallbladder disease. These receptors 26 Dec 2002 18:33 AR AR177-PH65-11.tex AR177-PH65-11.sgm LaTeX2e(2002/01/18) NUCLEAR RECEPTORS IN METABOLISM P1: IBC 269 could therefore partially underpin the high incidence of such diseases, which attains epidemic proportions and cannot be explained by genetic factors alone. Dysregulation of these receptors may also contribute to the syndromic clustering of several of these diseases in patients and families, under the name of syndrome X or the (pluri-)metabolic syndrome. The observation that these receptors seem to be dysregulated in many common diseases, and the fact that nuclear receptors are designed to respond to small molecules, made them excellent therapeutic targets for the development of preventive and therapeutic strategies for the treatment of these common disorders. The following sections present the role of metabolic nuclear receptors in adipocyte metabolism and insulin sensitivity, liver fat metabolism and reverse cholesterol transport, bile acid and intestinal lipid metabolism, and xenobiotic/endobiotic metabolism. PPARγ ’s Role in Adipocytes and Glucose Sensitivity A prime example of nuclear receptor involvement in metabolism is the pivotal role of PPARγ in adipocyte differentiation and lipid uptake. PPARγ , THE MASTER SWITCH OF ADIPOCYTE DIFFERENTIATION Several lines of evidence indicate that PPARγ is the master regulator of the differentiation and energy storage by adipocytes. The first line of support comes from molecular and cellular studies. The initial suggestion that PPARγ stimulated adipogenesis was based on the observation that overexpression of PPARγ in cultured cells was by itself sufficient to induce adipocyte differentiation (149). PPARγ was also shown to increase the expression of genes that promote fatty acid storage in adipocytes, such as fatty acid–binding protein (FABP) (150), lipoprotein lipase (LPL) (151), acyl-CoA synthase (152), phosphoenol pyruvate carboxykinase (PEPCK) (153), and perilipin. In contrast, PPARγ represses genes that induce lipolysis and release of fatty acids, such as the β 3-adrenergic receptor (β 3-AR) (154) and the cytokines leptin (155, 156) and TNF-α (157, 158). A number of excellent recent reviews have summarized these findings (159–161). A second argument supporting this hypothesis comes from human and animal genetic studies. Mice homozygous for a mutation in the PPARγ gene die in utero, secondary to a placental defect (59). Homozygous mice that survive to term by tetraploid rescue exhibit severe lipoatrophy (59). Interestingly, PPARγ +/− mice are resistant to obesity induced by high-fat diet and are more insulin sensitive, both on control (162) and high-fat diets (163). The resistance to obesity of PPARγ +/− mice is consistent with the primordial role of PPARγ in adipocyte differentiation and adipogenesis, with decreased preadipocyte differentiation resulting in an overall reduction in adipocyte mass and improved insulin sensitivity. Further genetic support for an important role of PPARγ in adipogenesis came from the characterization of patients with mutations in the human PPARγ gene. Subjects with the partial loss-of-function Pro12Ala mutation in the PPARγ 2-specific B 26 Dec 2002 18:33 270 AR AR177-PH65-11.tex AR177-PH65-11.sgm LaTeX2e(2002/01/18) P1: IBC FRANCIS ET AL. exon (164, 165) have a lower body mass index, greater insulin sensitivity, and an improved lipid profile (164, 166). The association between the hypomorphic Ala substitution and insulin sensitivity disappears when the data are corrected for the body mass index, suggesting a primary effect on body fat mass accretion (164). In contrast to the hypomorphic Pro12Ala allele, the rare Pro115Gln substitution renders PPARγ constitutively active through the modulation of the phosphorylation of PPARγ by MAPK on serine 114 (80, 81, 83). In keeping with the fact that PPARγ stimulates adipocyte differentiation, all carriers of this gain-of-function mutation are extremely obese and insulin resistant (81). A third independent argument supporting PPARγ ’s role in adipocyte differentiation is provided by pharmacological and physiological studies with various PPARγ ligands. Classical full PPARγ agonists such as the thiazolidinediones have been invariably shown to increase fat mass both in humans (167, 168) and rodents (78, 169, 170). PPARγ agonists furthermore redistribute adipose tissue from visceral to subcutaneous depots (167, 168, 171, 172) and induce the appearance of small, newly differentiated adipocytes at the expense of large, mature adipocytes (173–175). Whereas enhanced PPARγ activity is associated with an increase in adipose tissue mass, recent evidence has been accumulating that suboptimal PPARγ activation or PPARγ antagonism is neutral or even reverses weight gain. A good example of this is given by the partial PPARγ agonist FMOC-L-leucine, which binds and activates PPARγ with weaker affinity compared with full agonists (78). FMOC-L-Leu changes PPARγ structural conformation, triggering a distinct coregulator recruitment relative to agonist bound PPARγ . This differential coregulator recruitment contributes to the distinct biological effect of FMOC-L-Leu relative to full PPARγ agonists. In fact, FMOC-L-Leu is less adipogenic, but lowers glucose more effectively both in diet-induced and genetic models of insulin resistance (78). Similarly, other weak or partial PPARγ agonists, such as NC-2100 or MCC-555, also show little effect on adipocyte differentiation but have potent antidiabetic activities in vivo (176, 177). The PPARγ antagonists GW0072 (178), BADGE (179), and LG100641 (180) all inhibit adipocyte differentiation and adipogenesis in vitro. Furthermore, LG100641 was shown to blunt thiazolidinedione-induced target gene expression in 3T3-L1 adipocytes, yet stimulate glucose uptake (180). Inhibition of either PPARγ or RXR in vivo also improved insulin sensitivity and was associated with a decreased triglyceride content in white adipose tissue and skeletal muscle (181). These data indicate that decreasing or modulating PPARγ activity, rather than fully activating it, results in both decreased adipogenesis and improved glucose homeostasis. These findings are consistent with the effects of the partial loss of PPARγ function both in PPARγ +/− mice and humans with the Pro12Ala PPARγ substitution (162–165) and hint that it is possible to design selective PPARγ modulators that are better suited for the treatment of insulin resistance, compared with full PPARγ agonists. GLUCOSE HOMEOSTASIS: COMMUNICATION BETWEEN FAT, MUSCLE, AND PANCREAS On a whole-body level, glucose homeostasis involves several tissues. Although 26 Dec 2002 18:33 AR AR177-PH65-11.tex AR177-PH65-11.sgm LaTeX2e(2002/01/18) NUCLEAR RECEPTORS IN METABOLISM P1: IBC 271 mainly expresssed in adipose tissue, PPARγ has a major impact on the other tissues involved in glucose metabolism. Adipose tissue integrates energy homeostasis and metabolic control It is well established that adipose tissue is required for proper glucose homeostasis. Absence of adipose tissue, such as occurs in lipoatrophy and lipodystrophy in both mice and humans, leads to severe insulin resistance (182–184). The observation that classical PPARγ agonists increase fat mass along with improving glucose control supports the notion that adipose tissue mediates some of their beneficial effects on glucose homeostasis. Furthermore, the reduction in the relative amount of visceral fat and the appearance of increased numbers of more insulin-responsive small adipocytes and reduction in large mature adipocytes are changes associated with improved insulin-mediated glucose disposal (181, 185). White adipose tissue secretes a large number of signaling factors that impact on energy, lipid, and glucose homeostasis. Free fatty acids are a major group of such adipocyte-derived signaling molecules. A reduction in circulating free fatty acids is an early consequence of PPARγ activation and precedes the decrease in glucose and triglyceride levels (186). PPARγ -mediated induction of genes favoring lipid storage in adipose tissue promotes fatty acid repartitioning (also termed fatty acid steal) toward fat rather than muscle (187). Often, the same genes involved in lipid metabolism that are upregulated in adipose tissue by PPARγ are either not modulated (151, 188, 189) or decreased in skeletal muscle (186). Consistent with this finding, the level of insulin sensitization upon PPARγ activation is tightly correlated with a diminution in lipid accumulation in skeletal muscle (190). All these data clearly underscore the physiological impact of fuel partitioning between fat and muscle on insulin sensitivity and triglyceride homeostasis (191–196). Adipocytokines are other signaling factors that could mediate the effects of PPARγ on insulin sensitivity. Expression of tumor-necrosis factor-α (TNF-α) is induced in obesity, which results in a reduction of insulin-stimulated glucose uptake (197). Interference with TNF-α signaling, in mice with mutations either in the TNF-α gene or in both TNF-α receptors (p55 and p75), protects them against the development of obesity and insulin resistance (198–200). Activation of PPARγ attenuates TNF-α expression (157, 189), and this could contribute to the improvement in glycemic control (158). Leptin (201) and resistin (202), both secreted proportionally with adipose tissue mass, also belong to this class of signaling molecules repressed by PPARγ . The relevance of leptin and resistin repression as mediators of PPARγ activity remains unclear. Certain studies reported that PPARγ activators stimulate rather than decrease resistin expression (203), and most in vivo studies indicate that leptin increases insulin sensitivity (204, 205). Unlike the adipocytokines discussed above, adiponectin levels are inversely related to the amount of fat (206, 207). The adiponectin gene maps to chromosome 3q27, a locus that is associated with susceptibility to diabetes (208) and the metabolic syndrome (209). Reduced adiponectin concentrations are associated with insulin resistance in humans (210) and monkeys (211), whereas adiponectin administration enhances 26 Dec 2002 18:33 272 AR AR177-PH65-11.tex AR177-PH65-11.sgm LaTeX2e(2002/01/18) P1: IBC FRANCIS ET AL. insulin sensitivity including hepatic insulin action in mouse models of obesity (212, 213). PPARγ activation increases adiponectin production in humans (214) and insulin-resistant mice (212), which could contribute to the insulin-sensitizing effects of PPARγ ligands. Other mechanisms and tissues involved in glucose homeostasis The role of PPARγ in the control of glucose homeostasis likely extends beyond its primary effects in adipose tissue. Thiazolidinediones have been reported to induce the expression of insulin receptor substrate (IRS)-1 (215), IRS-2 (216), the p85 subunit of the phosphatidylinositol 3-kinase (PI3-kinase) (217), and the Cbl-associated protein (CAP) (218, 219). These results suggest that PPARγ might be more tightly coupled to insulin signaling than originally speculated. Direct effects of thiazolidinediones on insulin-stimulated glucose uptake in L6 myotubes (220, 221) and in cultured human skeletal muscle cells (222, 223) have been reported. Consistent with this, PPARγ activation was recently shown to repress the expression of muscle pyruvate dehydrogenase kinase 4 (PDK4) (186), an enzyme that inhibits glucose oxidation and whose expression negatively correlates with insulin-stimulated glucose uptake (224). Finally, increased activity of the glucose transporter GLUT4 and of PI-3-kinase has also been reported after thiazolidinedione treatment of muscle cells in vitro (221, 222, 225, 226). PPARγ is also expressed in pancreatic β-cells (66). Whereas activation of PPARγ does not acutely improve insulin secretion in pancreatic islets (66), evidence is accumulating that it protects β-cells from failure and apoptosis, although this issue is controversial (227). Development of type 2 diabetes is associated with intracellular accumulation of triglycerides in β-cells. Thiazolidinediones have been reported to increase fatty acid oxidation in β-cells, thereby delaying β-cell failure (228–232), and even to enhance pancreatic growth (233). In addition, a PPRE has been identified in the promoter region of the GLUT2 (234), and the glucokinase genes (235), proteins responsible for glucose transport and metabolism in β-cells. PPARγ -mediated enhancement of the pathway modulated by these proteins would stimulate the initial steps leading to insulin release. All these observations support the hypothesis that PPARγ -mediated restoration or protection of β-cell function could contribute, at least in part, to the control of glucose homeostasis (236, 237). Whereas some reports have shown that thiazolidinediones reduce hepatic glucose production (238–240), others have reported no effect (241). Furthermore, it is still unclear whether this effect on glucose production is a direct action on the liver or a consequence of a general increase in insulin action. Because most of the above studies on the effects of PPARγ in muscle, pancreas, and liver are based on results generated by using PPARγ ligands, and therefore potentially compromised by the specificity of the ligand, we expect that tissue-specific deletions of the PPARγ gene in the near future will shed more light on its respective roles in these tissues. The combined effects of PPARγ on fat and glucose metabolism are summarized in Figure 1. 26 Dec 2002 18:33 AR AR177-PH65-11.tex AR177-PH65-11.sgm LaTeX2e(2002/01/18) NUCLEAR RECEPTORS IN METABOLISM P1: IBC 273 Figure 1 Effects of PPARγ activation on fat and glucose metabolism. PPARγ induces adipocyte differentiation and numerous adipocyte genes involved in fat deposition. Fatty acids derived from hydrolysis of triglyceride-rich lipoproteins are redirected toward adipose tissue rather than skeletal muscle, which increases glucose metabolism in the muscle. PPARγ also induces the expression of genes involved in glucose uptake and insulin signaling in both adipose tissue and muscle, as well as a modulation of fat-derived signaling molecules that could affect glucose processing in the periphery. Finally, PPARγ seems to protect pancreatic β-cells against the intracellular triglyceride accumulation that is often associated with type 2 diabetes, and improves β-cell function. Abbreviations are defined in the footnote or in the text. Nuclear Receptor Control of Triglyceride Metabolism, HDL Formation, and Reverse Cholesterol Transport The liver is another crucial organ in the maintenance of fat and carbohydrate homeostasis and is the single most important organ in cholesterol metabolism. The nuclear receptors PPARα and LXR control multiple steps in the hepatic metabolism 26 Dec 2002 18:33 274 AR AR177-PH65-11.tex AR177-PH65-11.sgm LaTeX2e(2002/01/18) P1: IBC FRANCIS ET AL. of fats, the synthesis and catabolism of lipoproteins, and critical hepatic and extrahepatic steps in the synthesis of high-density lipoprotein (HDL) and the reverse cholesterol transport pathway. PPARα AND TRIGLYCERIDE METABOLISM The primary PPAR subtype expressed in liver is PPARα. Binding to PPARα by fatty acid, eicosanoid, and fibrate drug ligands leads to activation of numerous genes involved in the uptake and β-oxidative catabolism of fatty acids (39, 40, 42). In addition to inducing all the genes encoding enzymes of the classical β-oxidation pathway (e.g., acyl-CoA oxidase, very-long- and medium-chain acyl-CoA dehydrogenase, 3-ketoacyl-CoA thiolase) (242), PPARα also activates the genes necessary for cellular uptake of fatty acids (fatty acid transport protein) (194) and their initial derivatization for entry into the β-oxidation pathway (acyl-CoA synthetase) (194, 243). PPARα also stimulates fatty acid oxidation by these enzymes in the other tissues in which it is expressed, including heart, muscle, and kidney (244, 245). Increased diversion of fatty acids into β-oxidation decreases the availability of fatty-acyl CoA substrates for triglyceride synthesis and, therefore, very low density lipoprotein (VLDL) secretion by the liver. In addition to limiting substrate availability for triglyceride synthesis, PPARα has been reported to inhibit expression of apolipoprotein (apo) C-III (246, 247), a protein that inhibits both the triglyceride-hydrolyzing action of lipoprotein lipase and the uptake of triglyceride-rich lipoprotein remnants (248, 249). Although active in inhibiting apo C-III in rodents, the ability of PPARα agonists to lower apo C-III in humans remains controversial (250, 251). PPARα and γ agonists may also decrease triglyceride levels by increasing the expression of lipoprotein lipase (LPL) in the liver (PPARα) (151) and in adipocytes (151), skeletal muscle (252) (PPARγ ), and macrophages (PPARα and γ ) (253, 254). Conversely, PPARα ligands have been reported to decrease LPL activity in the heart (188, 255). How much of the hypotriglyceridemic effect of PPARα ligands can be ascribed to direct versus indirect effects (via decreased apo C-III) on LPL is not known. Recently, LXRα activation has also been shown to increase LPL expression in the liver and adipocytes, suggesting an additional mechanism of nuclear receptor-dependent LPL and triglyceride metabolism (256). NUCLEAR RECEPTOR EFFECTS ON HDL FORMATION The critical importance of nuclear receptors on lipid metabolism is also demonstrated by the role of these receptors in all three major mechanisms of formation of HDL particles. HDL levels are strongly and inversely correlated with risk for atherosclerotic vascular disease, with several epidemiologic studies from various sites worldwide indicating HDL is the strongest predictor of this risk [reviewed in (257)]. The realization of the critical importance of HDL levels, in addition to those of the atherogenic lipoproteins, in atherosclerosis risk has led to a vigorous search for novel agents to raise HDL levels clinically. HDL is thought to be protective for numerous reasons (258), the chief among these being stimulation of the return of cholesterol to the liver for excretion in bile, 26 Dec 2002 18:33 AR AR177-PH65-11.tex AR177-PH65-11.sgm LaTeX2e(2002/01/18) NUCLEAR RECEPTORS IN METABOLISM P1: IBC 275 or reverse cholesterol transport (259). Numerous transgenic animal models, while not identifying the major protective action of HDL, have demonstrated the protective effects of overexpressing human apoA-I and raising HDL levels in decreasing the formation of atherosclerotic lesions (260–263). Infusion of a proapoA-I liposome complex into hypercholesterolemic human patients was found to increase fecal steroid excretion, indicating that raising HDL levels does indeed increase total reverse cholesterol transport and cholesterol excretion (264). Enhancement of apoA-I and apoA-II synthesis by PPARα Synthesis of apo A-I and apo A-II, the two major proteins of HDL, by the liver and intestine is the first step in HDL particle formation. Increased apo A-I and apo A-II synthesis by the liver occurs in response to fibrate-induced PPARα activation of the human apolipoprotein A-I and A-II genes (265–268). In a recent study, rabbits that express the human apo A-I transgene along with its PPAR response elements were shown to have an increase in human apo A-I mRNA and mass in response to fenofibrate treatment in the absence of any peroxisome proliferative effect of the fibrate (269). Delivery of redundant surface components of triglyceride-rich lipoproteins to HDL The second mechanism for increasing HDL-cholesterol concentrations in plasma is the delivery of redundant surface phospholipid and apolipoprotein components of VLDL and chylomicrons onto HDL during hydrolysis of triglyceride-rich particles by lipoprotein lipase (270). Hypertriglyceridemia of any cause can lead to lowering of HDL cholesterol owing to cholesteryl ester transfer protein (CETP)-mediated transfer of triglyceride to HDL from triglyceride-rich particles and displacement of HDL cholesterol (271). Enhancement of triglyceride hydrolysis by PPARαinduced reduction in apo C-III synthesis and increase in LPL synthesis and activity decreases triglyceride levels, increases the transfer of triglyceride-rich particle surface components to HDL, and reduces the replacement of HDL cholesterol by triglyceride (270, 272). The overall contribution of this mechanism of HDL formation to total HDL cholesterol levels is currently unknown. Apolipoprotein-mediated cellular lipid efflux and ABCA1 The third and ratelimiting mechanism of HDL formation is the removal of cellular phospholipids and cholesterol by lipid-free or lipid-poor HDL apolipoproteins (273). The importance of this pathway was highlighted by the discovery that, despite normal synthesis of HDL apolipoproteins (274, 275), cultured cells isolated from patients with the low HDL syndrome Tangier disease failed to release both phospholipids and cholesterol to apo A-I (276, 277). The extremely low HDL levels in Tangier disease suggest that apolipoprotein-mediated cellular lipid efflux, as opposed to apolipoprotein synthesis, retrieval of redundant triglyceride-rich lipoprotein components, or passive cholesterol efflux down a concentration gradient to a lipid-containing surface (278), was the most critical determinant of HDL particle formation. The subsequent discovery of the gene defect in Tangier disease and familial HDL deficiency, mutations in the ATP-binding cassette transporter AI (ABCA1) (279–284), and the discovery of the regulation of ABCA1 by nuclear receptors has now generated 26 Dec 2002 18:33 276 AR AR177-PH65-11.tex AR177-PH65-11.sgm LaTeX2e(2002/01/18) P1: IBC FRANCIS ET AL. a huge interest in the study of nuclear receptor–dependent mechanisms to raise HDL levels for the treatment of atherosclerosis. Members of the ABC transporter superfamily are involved in transmembrane transport of a large variety of substrates. They couple the energy provided by ATP hydrolysis to the transport of substrates across membranes. The ABCA1 transporter is localized to the plasma membrane, early and late endosomal, lysosomal, and possibly Golgi compartments of cells (282, 285, 286). This transporter actively mediates either phospholipid alone (287–289) or both phospholipid and cholesterol delivery (282, 290, 291) from cells to apoA-I from both intracellular and plasma membrane lipid sources (292). The widespread expression of human ABCA1 in numerous tissues (293, 294), including the liver and even the vascular endothelium (295), strongly suggests the likelihood that apoA-I-mediated lipid efflux via ABCA1 occurs in numerous tissues in addition to macrophages. We have recently found that apoA-I mediates the depletion of cholesterol and phospholipids from cultured primary hepatocytes, generating HDL particles and decreasing the secretion of VLDL (G. Francis & R. Lehner, unpublished observations). These results suggest that, in addition to peripheral (non-hepatic) cells, the liver itself is a source of lipids for HDL formation that may then be redirected back to the liver for excretion in the reverse cholesterol transport pathway. The upregulation of ABCA1 for HDL formation, regardless of the main tissue supplying these lipids, by nuclear receptors is therefore a promising direction to pursue in the development of new anti-atherosclerotic therapies. The expression of ABCA1 is tightly regulated by the cellular content of cholesterol, through oxysterol-dependent activation of LXR (296–299). An LXR response element has been identified in the human ABCA1 promoter that binds both LXRα and LXRβ and mediates transcriptional induction of this promoter by LXR and RXR ligands (296, 300). Therefore, LXR/RXR-stimulated cholesterol efflux from macrophages, mediated through the induction of ABCA1 expression, is likely responsible for the dramatic reduction in atherosclerosis observed in apoE −/− mice treated with RXR ligands (301). Other nuclear receptors, including PPARα and PPARγ , activate ABCA1 expression indirectly via enhanced transcription of LXRα (302–304). Transplantation of PPARγ -null bone marrow into LDL receptor −/− mice resulted in a significant increase in atherosclerosis, suggesting that regulation of LXR and ABCA1 by PPARγ has an important protective effect in vivo (304). Targeted PPARγ deficiency resulted in a marked reduction of LXRα expression and macrophage cholesterol efflux to HDL (254, 304, 306). These in vivo data underscore the potential usefulness of synthetic LXR, PPAR, or RXR agonists in the prevention or treatment of atherosclerosis as recently suggested by two studies (301, 305, 305a). A selective PPARβ/δ agonist has also been reported that enhances expression of ABCA1, raises HDL, and improves other lipid parameters in insulin-resistant monkeys (52). Consistent with a potential beneficial effect of pharmacological activation of LXR/RXR is the fact that the ABCA1 promoter is only suboptimally activated by oxysterols that are relatively abundant in foam cells (300). In addition, ABCA1 expression is downregulated rapidly upon HDL or apoA-I-mediated removal of 26 Dec 2002 18:33 AR AR177-PH65-11.tex AR177-PH65-11.sgm LaTeX2e(2002/01/18) NUCLEAR RECEPTORS IN METABOLISM P1: IBC 277 lipids from cultured cells (282, 293). Synthetic LXR agonists are being developed that may have the ability to increase ABCA1-dependent lipid mobilization to apoA-I and increase HDL levels clinically, and supercede the need for high cell oxysterol content to turn on ABCA1 (91, 95, 307, 308). One concern about LXR agonists has been their propensity to increase triglyceride levels through activation of SREBP-1c (95, 309) and induction of fatty acid synthase (308, 310). To date, the mouse models of atherosclerosis used have shown an ability of LXR and other nuclear receptors agonists to increase ABCA1 expression and decrease atherosclerosis, but consistent effects on HDL and triglyceride levels have not been found (301, 305, 311). Overexpression of the human ABCA1 transgene in normal mice led to an increase in HDL and decrease in atherosclerosis (312), providing further promise that enhancement of ABCA1 expression is a useful route to pursue in the development of anti-atherosclerotic therapies. Nuclear receptor agonists that specifically enhance ABCA1 expression without inducing hypertriglyceridemia are currently being pursued. Other mechanisms of enhancing ABCA1 expression, such as the use of cyclic AMP-dependent pathways (313, 314), may also prove useful as novel means to raise HDL. Although mutations of the ABCA1 gene are the major cause of both Tangier disease and familial HDL deficiency, another member of the ABC transporter superfamily, the human white gene (ABCG1, mouse ABC8), may also have an important role in HDL and apoA-I-dependent cellular lipid efflux (315). As seen with ABCA1, it has been shown that mRNA levels of both the murine ABC8 protein and its human homolog are induced by LXR- and RXR-specific ligands in an LXR-dependent mechanism (316). Another important way LXR could contribute to cholesterol efflux is by mediating the LXR-ligand dependent activation of apolipoprotein E (apoE) expression in macrophages and adipose tissue (317). LXR/RXR heterodimers regulate apoE transcription directly in vivo, through interaction with a conserved LXR-response element (RE) present in two enhancers of the apoE gene. Because basal apoE expression is preserved in LXR −/− mice, it is suggested that LXR mediates principally lipid (oxysterol)-inducible expression of apoE (317). It was recently shown that the entire apoE/C-I/C-IV/C-II gene cluster was subject to direct regulation by LXR in murine and human macrophages (318). LXRα has been shown to be specifically induced during human macrophage differentiation (319). Moreover, the human LXRα gene, but not the LXRβ, is regulated directly by both LXRα/RXR and LXRβ/RXR heterodimers, which bind to a single LXR-RE in the human LXRα gene promoter. This autoregulation increases the expression of LXRα target genes ABCA1, ABC8, and apoE in response to oxysterols (320–322). This LXRα autoregulatory loop provides a mechanism for efficiently amplifying the effects of oxysterols in macrophages and promoting reverse cholesterol transport. ADDITIONAL NUCLEAR RECEPTOR EFFECTS ON REVERSE CHOLESTEROL TRANSPORT In addition to enhancing ABCA1 and apoE expression, nuclear receptors have also been implicated in the expression of other proteins in the reverse cholesterol 26 Dec 2002 18:33 278 AR AR177-PH65-11.tex AR177-PH65-11.sgm LaTeX2e(2002/01/18) P1: IBC FRANCIS ET AL. transport pathway that facilitate the transport of tissue-derived cholesterol back to the liver for excretion in bile. Following the delivery of phospholipids and probably some cholesterol to apoA-I via ABCA1, the nascent HDL particle dissociates from ABCA1 (323) and circulates in plasma, where it is converted into larger HDL particles by deriving additional lipids from cells via passive cholesterol efflux (324), the LPL-dependent hydrolysis of triglyceride-rich lipoproteins, and transfer of lipids from triglyceride-rich lipoproteins via phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP). It has been reported that FXR activated by bile acids induces the PLTP gene through FXR/RXR heterodimer binding to the PLTP gene FXR response element (325). PLTP may also be activated by PPARα, which may explain a portion of the HDL-raising effects of fibric acid derivatives (326). CETP mediates the transfer of HDL cholesteryl esters to apoB-containing particles for return to the liver, and in exchange it receives triglycerides from these particles (327). This modification of HDL by CETP makes the HDL more susceptible to hydrolysis by hepatic lipase at the hepatocyte surface, an important component of the regeneration of small HDL particles and free apoA-I that can then recirculate in the reverse cholesterol transport pathway. In this way, CETP likely represents an anti-atherogenic protein. The CETP gene has been shown to be upregulated in response to hypercholesterolemia (328). Luo et al. showed that both LXRα and LXRβ transactivate the CETP promoter in a sterol-dependent manner (329) and hence coordinately regulate the CETP-mediated delivery of HDL cholesterol esters to the liver. In addition, LXRα mRNA was found to be markedly induced in differentiated 3T3-L1 adipocyte cells, another cell type capable of producing CETP (329, 330). The induction of LXRα during adipocyte differentiation is correlated with the appearance of sterol induction of the CETP promoter in differentiated adipocytes (329). The orphan nuclear receptor LRH-1 also transactivates the CETP promoter by binding to a proximal promoter element distinct from the LXRα site (130) and potentiates the sterol-dependent regulation of the wild-type CETP promoter by LXR. SHP abolishes this potentiating effect of LRH-1, but not the basal transactivation of the CETP promoter (130). The final uptake of cholesterol carried on HDL by the liver is mediated through the concerted actions of scavenger receptor B-I (SR-BI) and hepatic lipase. These players in reverse cholesterol transport are also affected by nuclear receptors. Previously we showed a decrease in hepatic lipase activity by activation of PPARα with fenofibrate (331). Furthermore, our laboratory recently established that the expression of the SR-BI gene is induced in the liver and the ovary, two tissues that utilize HDL-derived cholesterol, by LRH-1 (G. Schoonjans, E. Fayard & J. Auwerx, unpublished data), thereby contributing to reverse cholesterol transport. Although the role of SR-BI in mediating cholesterol efflux to HDL from peripheral cells remains controversial, some evidence suggests that SR-BI is expressed in atherosclerotic tissue macrophages and is increased in cultured macrophages in response to both PPARα and PPARγ ligands (332). The effects of nuclear receptors on several mediators of reverse cholesterol transport are indicated in Figure 2. 26 Dec 2002 18:33 AR AR177-PH65-11.tex AR177-PH65-11.sgm LaTeX2e(2002/01/18) NUCLEAR RECEPTORS IN METABOLISM P1: IBC 279 Figure 2 Nuclear receptor regulation of reverse cholesterol transport. Direct regulation of LXR by oxysterols, LXR autoregulation, indirect regulation of LXR via PPARα and PPARγ , and possibly LXR-independent regulation by PPARβ/δ induces ABCA1 expression in macrophages and probably in other tissues including the liver. ABCA1 mediates the delivery of lipids to apoA-I for HDL particle formation. LXR also triggers the expression of ABC8 and apoE in macrophages, and CETP, CYP7A1, and CYP8B1 in the liver. FXR increases PLTP expression, which along with CETP facilitates the conversion of HDL to better substrates for hepatic lipase and SR-BI-dependent cholesterol uptake. PPARα increases apoA-I expression and inhibits hepatic lipase expression by the liver. BA, bile acids; C, cholesterol; CE, cholesteryl esters; PL, phospholipid; OxC, oxysterols; HL, hepatic lipase; LDLr, LDL receptor. For Figures 2 to 4, nuclear receptors are represented in ovals, target genes for nuclear receptors are represented in boxes and are in italics; fine continuous lines represent regulatory pathways, and dashed lines represent metabolic pathways. Inhibition is indicated by a, stimulation by →. A PARALLEL REVERSE CHOLESTEROL TRANSPORT PATHWAY Another potential pathway of reverse cholesterol transport involves the mitochondrial sterol-27 hydroxylase (CYP27). This enzyme, present in a number of tissues including liver, kidney, spleen, adrenal gland, heart, and found to some extent in all tissues studied 26 Dec 2002 18:33 280 AR AR177-PH65-11.tex AR177-PH65-11.sgm LaTeX2e(2002/01/18) P1: IBC FRANCIS ET AL. (333, 334), hydroxylates cholesterol to produce 27-hydroxycholesterol and the oxidized product, cholestenoic acid. These sterol metabolites are released into the circulation and transported to the liver where they are metabolized further into bile acids and excreted (335). Consistent with the hypothesis that CYP27 underpins a second reverse cholesterol transport pathway to the liver is the fact that 27-hydroxycholesterol is the most abundant form of hydroxycholesterol in the human circulation. Interestingly, CYP27-mediated hydroxylation of cholesterol is important for the generation of endogenous LXR agonists upon cholesterol loading of cells because it has been shown that 27-hydroxycholesterol (336) and cholestenoic acid (337) are agonists of LXRα and LXRβ. In vivo support of a role of CYP27 in reverse cholesterol transport can be found in the susceptibility of patients who carry a mutation in the CYP27 gene and suffer from the disease cerebrotendinous xanthomatosis, which results in premature atherosclerosis and the accumulation of sterols in multiple tissues (338, 339). Intriguingly, this phenotype is not entirely reflected in mice in which the CYP27 gene was targeted by homologous recombination (340). Nuclear Receptor Regulation of Bile Acid and Intestinal Lipid Metabolism The final step of reverse cholesterol transport—excretion of cholesterol and its metabolites in bile—and the initial steps of cholesterol absorption in the intestine are also closely integrated and under the control of nuclear receptors. In many species including humans, the conversion of cholesterol into bile acids is the only method of excreting cholesterol from the body. Bile acids are direct end products of cholesterol catabolism that are either excreted in the feces or reabsorbed in the intestine and returned to the liver in the pathway known as enterohepatic circulation. Efficient enterohepatic recirculation of bile acids is essential to maintain a sufficient bile acid pool. Bile acids not only stimulate the excretion of excess hepatic cholesterol into the bile, they are also essential for the solubilization and absorption of dietary cholesterol and fat-soluble vitamins. As there are significant differences between mice and humans in the composition of the bile acid pool, studies are necessary to determine whether the same mechanisms are operational in the different species. CONVERSION OF CHOLESTEROL INTO BILE ACIDS Cholesterol conversion into bile acids occurs via two different pathways: the classical and the alternative pathways, the end products of which are cholic acid and chenodeoxycholic acid. Three enzymes play major regulatory roles in these two pathways. Cholesterol 7α-hydroxylase (CYP7A1) is the rate-limiting enzyme in the classical pathway, whereas CYP27 is the first enzyme in the alternative pathway. Sterol 12α-hydroxylase (CYP8B1) in the classical pathway introduces a hydroxyl group at position 12 of the steroid nucleus of chenodeoxycholic acid, generating the more hydrophilic cholic acid. The ratio of cholic acid (hydrophilic) to chenodeoxycholic 26 Dec 2002 18:33 AR AR177-PH65-11.tex AR177-PH65-11.sgm LaTeX2e(2002/01/18) NUCLEAR RECEPTORS IN METABOLISM P1: IBC 281 acid (hydrophobic) therefore determines the relative hydrophobicity of the circulating bile acid pool. An altered ratio of cholic to chenodeoxycholic acid has been postulated to play a role in cholesterol gallstone formation because increased bile salt hydrophobicity promotes precipitation of cholesterol crystals (341). As a consequence of their importance in governing cholesterol homeostasis, the level of bile acids in the enterohepatic circulation is tightly controlled. Elevated cholesterol concentrations, often resulting from high dietary intake, result in an increased synthesis of oxysterols (342). Oxysterols bind to and activate LXRα, which stimulates the transcription of the mouse CYP7A1 (89, 90), resulting in increased bile acid synthesis and subsequent excretion of cholesterol. LXRα knockout mice fed a high-cholesterol diet fail to upregulate CYP7A1 expression and bile acid production, thereby accumulating cholesterol esters in their livers (343). In addition to LXRα, LRH-1, also identified as a CYP7A1 promoter-binding factor (CPF) (124), is a crucial competence factor facilitating basal CYP7A1 expression and subsequent regulation by LXRα-mediated sterol activation (127, 140, 141). Whether this regulation also occurs in humans has to be determined because no LXR response element has been identified in the human CYP7A1 gene. Consistent with a lack of LXR-dependent activation of the human CYP7A1 gene (343a), the LXR/RXR heterodimer failed to bind to the promoter region of the human CYP7A1 gene expressed in mice, leading to a lack of CYP7A1 transgene expression in response to dietary cholesterol (344). BILE ACIDS SUPPRESS THEIR OWN PRODUCTION In contrast to the stimulatory effects of LXR on bile acid synthesis in mice, bile acid–binding to FXR effectively inhibits bile acid synthesis by repressing CYP7A1 gene transcription (102–104). FXR acts indirectly on CYP7A1 transcription, since the CYP7A1 promoter contains no consensus FXR-binding sites (127). This repression is mediated through a mechanism involving the nuclear receptors LRH-1 and SHP (140, 141). As levels of bile acids increase, bile acid–binding activates FXR, turning on transcription of SHP. Elevated levels of SHP protein bind and inactivate LRH-1, leading to transcriptional repression of CYP7A1 (140, 141). The regulation of SHP mRNA levels by FXR was validated in an FXR-deficient mouse model, which failed to show activation of SHP in response to bile acids (345). In contrast to these studies in mice, in vivo studies in rats and in vitro studies in the human HepG2 cell line revealed that bile acid-activated FXR stimulates LRH-1 expression, which then directly represses both CYP7A1 and SHP expression in the liver (346). Moreover, the level of induction of SHP mRNA expression by bile acids in rat liver and (human) HepG2 cells was much less than that observed in mice (346). Hence, unlike in mice, the repression of CYP7A1 expression in rats and humans does not seem to involve SHP to a major extent (346). This suggests that species-dependent differences in promoter and cellular context are important in the control of CYP7A1 expression and that the relative expression levels of the nuclear receptors involved could be a further confounding factor. 26 Dec 2002 18:33 282 AR AR177-PH65-11.tex AR177-PH65-11.sgm LaTeX2e(2002/01/18) P1: IBC FRANCIS ET AL. Interestingly, the enzyme responsible for cholic acid synthesis, CYP8B1, is also a target gene for LRH-1 (126) and is downregulated by bile acids in a manner similar to CYP7A1, as deduced from the characterization of the FXR null mouse (345). Important species differences also exist in the CYP8B1 gene promoter region. In the rat, the CYP8B1 promoter contains two overlapping LRH-1 binding sites, whereas the human CYP8B1 contains only one. Recent reports suggest that SHP suppresses rat CYP8B1 promoter activity mainly via an interaction with LRH1 (347), whereas in the human gene, SHP exerts its repressive effect predominantly via HNF4-α (348). Because in the rat, HNF4-α is regulated by LRH-1 (349), interaction of SHP with LRH-1 may repress HNF4-α gene transcription. HNF4-α strongly activates human CYP8B1 promoter activity, whereas LRH-1 has much less effect (348). The CYP7A1 promoter has also been shown to bind HNF4-α in both rat (350) and human (351). Therefore, LXR, LRH-1, and HNF4-α regulate CYP7A1 and CYP8B1 transcription in a species- and gene-specific manner. BILE ACID EFFECTS ON ENTEROHEPATIC CIRCULATION The enterohepatic circulation of bile is critical for the intestinal absorption of nutrients, the biotransformation and excretion of cholesterol, and the clearance of potentially toxic xenobiotics and metabolites. When bound to bile acids, the FXR/RXR heterodimer stimulates the expression of the cytosolic ileal bile acid-binding protein (I-BABP) (352), which acts as a carrier protein facilitating the re-uptake of bile acids by the gut. Results from studies of FXR knockout mice provide convincing evidence that I-BABP is regulated in an FXR-dependent manner (345). Bile salts returning to the liver in portal blood are taken up by facilitated transport via at least two sinusoidal membrane bile salt transport proteins: the sodium taurocholate cotransporting polypeptide (NTCP), and the organic anion transporting polypeptide-1 (OATP-1). NTCP has been shown to mediate sodium-dependent uptake of all physiological bile salts (353), and FXR is involved in its bile acid-mediated downregulation as demonstrated in FXR ablated mice (345). More particularly, suppression of NTCP by bile acids involves the induction of the repressor SHP, which in this case interferes with the activity of the RXR/RAR heterodimer that controls the expression of the rat NTCP gene (354). Once in the hepatic cytosol, bile salts transit to the canalicular membrane, where they are actively extruded into the bile by the bile salt export pump (BSEP). BSEP, expressed exclusively in liver, is another member of the ABC protein superfamily and it was shown (by studies in FXR knockout mice) to be upregulated by FXR (345). Induction of the expression of the mouse and the human BSEP gene occurs through the direct interaction of the bile acid–activated FXR/RXR heterodimer, with a highly conserved element in the proximal BSEP promoter (113, 355). Nuclear receptor effects on bile acid metabolism and the enterohepatic circulation are summarized in Figure 3. NUCLEAR RECEPTORS AND INTESTINAL LIPID ABSORPTION The recent discovery of the role of ABCA1 in HDL formation has led to a huge increase in interest 26 Dec 2002 18:33 AR AR177-PH65-11.tex AR177-PH65-11.sgm LaTeX2e(2002/01/18) NUCLEAR RECEPTORS IN METABOLISM P1: IBC 283 Figure 3 Nuclear receptors and the regulation of bile acid metabolism and enterohepatic circulation. LXR, activated by oxysterols, limits hepatic cholesterol accumulation in rodents by stimulating CYP7A1, the rate-limiting step in bile acid synthesis. FXR, activated by bile acids, protects the liver against the toxic effects of excessive bile acid concentrations through an indirect mechanism involving induction of SHP. SHP attenuates further bile acid synthesis by inhibiting the action of LRH-1 on CYP7A1 and CYP8B1 expression. SHP also limits bile acid import into the liver by inhibiting NTCP expression. Moreover, FXR not only stimulates export of bile acids from the liver into the bile by directly increasing the expression of BSEP, it also stimulates the re-uptake of bile acids from the intestine by upregulating I-BABP in the gut. This process is termed enterohepatic circulation. in the role of this transporter in other tissues and in ABC transporters generally. Sterol absorption via ABC transporters in the gut is also under nuclear-receptordependent control. Administration of an LXR or RXR ligand to mice, in addition to upregulating CYP7A1 expression in the liver and ABCA1 in macrophages, was found to increase ABCA1 expression levels in all segments of the small intestine, and to decrease cholesterol absorption by the gut (298). This increased ABCA1 expression failed to occur in LXR α/β double knockout mice, confirming the importance of LXR in this response (298). Although suggestive of a direct role for ABCA1 in preventing cholesterol absorption, it is not known whether this occurs by active pumping of cholesterol back out of enterocytes into the intestinal lumen or by inhibiting cholesterol absorption indirectly. Two additional LXRdependent ABC transporters, ABCG5 and ABCG8, which were shown to be the genes mutated in the abnormality of plant sterol absorption sitosterolemia, are also targets for LXR regulation (356). The increased intestinal absorption and impaired 26 Dec 2002 18:33 284 AR AR177-PH65-11.tex AR177-PH65-11.sgm LaTeX2e(2002/01/18) P1: IBC FRANCIS ET AL. biliary secretion of plant sterols seen with mutations in either of these transporters suggests an active role for them in pumping these sterols back into the gut lumen from enterocytes, and into bile from hepatocytes. PXR/SXR and Metabolism of Endo- and Xenobiotics Normal homeostasis requires the detoxification and elimination of compounds of endogenous and exogenous nature that are harmful when accumulated to toxic levels. These potentially harmful compounds could be regrouped under the general term endo- and xenobiotics and include hormones, bile acids, carcinogens, prescription and non-prescription drugs, as well as certain molecules present in food and environmental pollutants. The metabolism and further elimination of these endo- and xenobiotics are accomplished by the concerted action of (a) the phase I oxidative CYP enzymes, (b) the phase II conjugating enzymes, and (c) the drug transporters in the enterohepatic axis (357, 358). Regulation of these detoxification pathways for endo- and xenobiotics occurs mainly through the transcriptional control of the hepatic CYP enzymes. These enzymes catalyze the conversion of endo- and xenobiotics to more polar derivatives that can be better cleared from the organism (359, 360). Among the CYP enzymes, the CYP3A and CYP2B enzymes are of particular medicinal relevance because they participate in the metabolism of 50–60% and 15–20% of clinically used drugs, respectively. They also contribute to the metabolism of plant metabolites and a variety of endogenous steroids, bile acids, and lipid metabolites (360). CYP expression varies strongly between people, but the factors causing this variation are at present unclear. This variation could influence the metabolism and elimination of drugs, environmental toxins, and carcinogens and may also contribute to other metabolic differences among individuals. MULTISTAGE DETOXIFICATION OF ENDO- AND XENOBIOTICS Whereas only a single CYP3A form exists in rabbits, multiple CYP3A forms are present in rat and mouse liver, and four CYP3A genes have been identified in humans (361, 362). Three arguments suggested that CYP3As could be PXR target genes. First, PXR and CYP3A genes are co-expressed in the liver, intestine, and kidney, tissues that are the primary sites of drug-metabolism (8). Second, many of the compounds known to induce CYP3A selectively in human, mouse, rat, or rabbit also activate the corresponding PXR by direct binding (107). Third, PXR was shown to bind to xenobiotic response elements previously identified in the human and rat CYP3A promoters and to induce CYP3A transcription in response to PXR activators (8, 105, 106, 108). The in vivo proof of the key role of PXR in the protection against toxic compounds was elegantly provided by the demonstration that PXR inactivation in the mouse, although not affecting basal expression of CYP3A, attenuates the induction of CYP3A in response to specific CYP3A inducers, such as dexamethasone or PCN, in both liver and intestine (111, 112, 116). Consequently, these PXR-null mice are no longer protected by PCN treatment against sedative 26 Dec 2002 18:33 AR AR177-PH65-11.tex AR177-PH65-11.sgm LaTeX2e(2002/01/18) NUCLEAR RECEPTORS IN METABOLISM P1: IBC 285 toxic compounds or the hepatotoxic bile acid LCA. In contrast, hepatic expression of a constitutively active human PXR transgene results in upregulation of CYP3A gene expression and subsequent protection against toxic compounds (116). In addition to CYP3A, PXR was also reported to control the expression of additional phase I enzymes, such as CYP2C8 and CYP2C9 (363–365) and CYP2B6 (363, 366), both involved in the metabolism of a number of clinically important drugs (367) and the phase II glutathione S-transferase A2 (368). In parallel to PXR’s ability to induce phase I and II detoxifying enzymes, several PXR agonists have been shown to induce expression of certain endoand xenobiotic transporters. Uptake of compounds by hepatic/intestinal sinusoidal transporters is critical for the entry of some chemicals into hepatocytes, where they are biotransformed and subsequently excreted through the intestinal lumen. The multidrug resistance 1 (MDR1) gene encodes the intestinal drug transporter P-glycoprotein (ABCB1), a member of the ABC transporter family, and plays an important role in the elimination of many endo- and xenobiotics by transporting hydrophobic compounds outside of the cells. It hence functions as an efflux pump, actively transporting substances back into the intestinal lumen. The antibiotic rifampicin was known to induce the intestinal P-glycoprotein (369). This effect is mediated through PXR, which binds a DR4 nuclear receptor response element in the MDR1 promoter (365, 370). The multiple resistance protein 2 (MRP2, ABCC2) is another member of the ABC family of transporter proteins recently shown to be regulated by PXR (371, 372). This bile canalicular membrane transporter is a critical regulator of bile flow and biliary drug excretion and is involved in the export of organic anions, various conjugated compounds, glutathione, and xenobiotics such as some anticancer drugs (373–378). For this reason, it is thought to be responsible for drug resistance. MRP2 is also expressed in the kidney, jejunum, and ileum, where it could also be involved in the excretion of toxic compounds from the body (374). Moreover, in addition to being induced by PXR, MRP2 is also regulated by two other nuclear receptors, i.e., CAR and FXR, which like PXR bind to the same response element on the MRP2 promoter (372). The Na+-independent organic anion transporter 2 (OATP-2) is a liver sinusoidal transporter that mediates the hepatic uptake of a broad array of endogenous and exogenous compounds (379–381). Data generated using PXR knockout mice suggest that PXR is required for induction of OATP2 by PCN (111). Furthermore, the rat OATP2 has been identified as a direct target gene for PXR, which interacts with three main response elements on the promoter (382). BILE ACID DETOXIFICATION Despite their beneficial function, excessive concentrations of bile acids are potentially toxic when accumulated in the body. This is particularly true for lithocholic acid (LCA), a hydrophobic secondary bile acid that is primarily formed in the intestine by the bacterial 7α-dehydroxylation of chenodeoxycholic acid. LCA causes cholestasis, a pathogenic state characterized by decreased bile flow and the accumulation of bile constituents in the liver and 26 Dec 2002 18:33 286 AR AR177-PH65-11.tex AR177-PH65-11.sgm LaTeX2e(2002/01/18) P1: IBC FRANCIS ET AL. Figure 4 The endo- and xenobiotic- responsive nuclear receptor PXR controls bile acid detoxification. PXR exerts a hepatoprotective function against bile acid (BA) accumulation by inhibiting CYP7A1 expression. In addition, PXR induces OATP-2, which facilitates the uptake of toxic secondary bile acids, such as LCA, to the liver. There, LCA activates PXR, which favors the catabolism of toxic compounds (such as LCA) by inducing the expression of CYP3A and CYP2B enzymes. blood. The potentially harmful effects of LCA and other bile acids are attenuated by PXR-controlled hepatic detoxification pathways, which render the bile acids more hydrophilic and facilitate their excretion in the feces or urine. PXR inhibits production of additional bile acids by inhibiting CYP7A1, and induces OATP2, a sinusoidal membrane transporter, which increases the uptake of LCA and other bile acids from sinusoidal blood into the hepatocyte. In the hepatocyte, LCA is then hydroxylated by CYP3A11, another PXR target (112), or by other CYP3A subfamily members. The importance of this regulation by bile acids such as LCA, which act as functional ligands for PXR, highlights the intricate control pathway preventing bile acid toxicity (111–113). These effects of PXR are summarized in Figure 4. DRUG-DRUG INTERACTIONS The regulation of hepatic CYP enzymes is implicated not only in drug metabolism and detoxification but also in drug-drug interactions. Because CYP3A and CYP2B enzymes can recognize a large variety of 26 Dec 2002 18:33 AR AR177-PH65-11.tex AR177-PH65-11.sgm LaTeX2e(2002/01/18) NUCLEAR RECEPTORS IN METABOLISM P1: IBC 287 pharmaceutical substrates, compounds that are inducers of these enzymes through activating PXR could affect the metabolism and clearance of any co-metabolized drugs that are substrates for the enzymes. A good example is given by St. John’s wort, an herbal product popular to treat depression, which has been shown to increase the metabolism and consequently the inactivation of various drugs including oral contraceptives, immunosuppressants such as cyclosporin, and HIV protease inhibitors such as indinavir (383–387). One of the constituents of St. John’s wort, hyperforin, was shown to be a potent PXR ligand, which induces the expression of CYP enzymes that metabolize these particular drugs when co-administered (388). INTERPLAY BETWEEN NUCLEAR RECEPTORS IN ENDO- AND XENOBIOTIC DETOXIFICATION Whereas PXR can induce CYP2B, activation of CYP2B subfamily members by phenobarbital and phenobarbital-like inducers was previously shown to be mediated by another nuclear receptor, the constitutive androstane receptor (CAR) (389, 390). CAR is an RXR heterodimer partner that activates gene transcription constitutively (391). Several groups have recently demonstrated that PXR and CAR can cross-regulate their respective target genes CYP2B and CYP3A (366, 392, 393). This cross-regulation is explained by the fact that both PXR/RXR and CAR/RXR can bind and activate common response elements in some of the CYP3A and CYP2B genes (106, 111, 392–394) and suggests that interplay between these receptors might be an important factor in the regulation of the xenobiotic-metabolizing enzymes and constitutes an efficient and partially redundant safety mechanism. Similarly to PXR, CAR also exhibits clear speciesdependent ligand specificity (107, 395, 396). CONCLUSIONS The activation of nuclear receptors by metabolites underlies most of the genetic responses controlling fat, glucose, cholesterol, bile acid, and xenobiotic/endobiotic metabolism. PPARγ is the major receptor mediating the response of adipocytes to energy loading. PPARα regulates the catabolism of fatty acids and impacts several steps in the reverse cholesterol transport pathway. LXR regulates many aspects of cholesterol removal from tissues, its conversion into bile acids for excretion, and its absorption in the gut. FXR regulates the re-uptake of bile acids in the enterohepatic circulation, plus the feedback inhibition of bile acid synthesis by bile acids through an indirect mechanism involving the nuclear receptors LRH-1 and SHP. Finally, PXR/SXR exerts a protective function against the harmful effects of excessive levels of secondary bile acids and xenobiotic compounds. Although our review aimed to summarize current knowledge about the regulatory activities of these metabolic receptors, it also points to a complex cross-regulation that exists between these receptors to control lipid, glucose, cholesterol, and bile acid homeostasis. Further studies will definitely be required before the full importance of these vital and complex regulatory circuits is fully understood. Furthermore, because nuclear receptor dysregulation is a feature of many of the most common 26 Dec 2002 18:33 288 AR AR177-PH65-11.tex AR177-PH65-11.sgm LaTeX2e(2002/01/18) P1: IBC FRANCIS ET AL. diseases today and because ligand-activated transcription factors are excellent drug targets, such studies will definitely lead to novel prevention and treatment strategies for many metabolic disorders. ACKNOWLEDGMENTS Work in the laboratories of the authors is supported by grants of CIHR (MOP12660), CNRS, INSERM, ULP, Hôpital Universitaire de Strasbourg, HFSP (RG0041/1999-M), NIH (1P01 DK59820-01), and the EU (GLG1CT-1999-00674 and GLRT-2001-00930). FP was supported by a postdoctoral fellowship from the Canadian Institutes of Health Research. GAF is a Scholar of the Alberta Heritage Foundation for Medical Research. EF received support from ARC (Association pour Recherchesur le Cancer) JA is a research director with CNRS. The Annual Review of Physiology is online at http://physiol.annualreviews.org LITERATURE CITED 1. Maglich JM, Sluder A, Guan X, Shi Y, McKee DD, et al. 2001. Comparison of complete nuclear receptor sets from the human, Caenorhabditis elegans and Drosophila genomes. Genome Biol 2(8): RESEARCH0029 2. Robinson-Rechavi M, Carpentier AS, Duffraisse M, Laudet V. 2001. How many nuclear receptors are there in the human genome? Trends Genet. 17:554– 56 3. 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