FXR acts as a bile acid sensor and is involved in a negative feedback regulation controlling bile acid production (reviewed in Edwards et al., 2002). FXR was initially cloned as an RXR-interacting protein called RIP14 (Seol et al., 1995). Its expression is restricted to adrenal cortex, intestine, colon, kidney, and liver. FXR forms heterodimers with RXR to bind to DNA response elements (FXREs), most often consisting of two hexamers (GGGTCA or close derivatives), organized in an inverted palindromic configuration, and spaced by one nucleotide.
Initially proposed as a receptor of farnesol metabolites, FXR was shown to bind and be activated by physiological concentrations of free and conjugated bile acids that are the end-products of the neutral and acidic bile acid biosynthetic pathway: chenodeoxycholic acid, lithocholic acid, and deoxy-cholic acid (Makishima et al., 1999; Parks et al., 1999; Wang et al., 1999). FXR senses bile acids and responds by inhibiting further bile acid synthesis, as illustrated in FXR null mice. These mice, which have no overt phenotype except increased bile acid levels in the blood, cannot sustain a cholic acid-enriched diet. They suffer from a severe wasting syndrome with hypothermia, and around 30% of them die by day 7 on such a diet (Sinal et al., 2000).
FXR acts at multiple levels of bile acid metabolism. First, it negatively regulates CYP7A whose expression positively controls the neutral pathway of bile acid synthesis (Goodwin et al., 2000; Lu et al., 2000). Second, it decreases the expression of the sodium taurocholate cotransporting poly-peptide (NTCP), which mediates the uptake of bile acids in hepatocytes along the enterohepatic cycle (Denson et al., 2001; Sinal et al., 2000). These two inhibitions of gene expression occur via an indirect mechanism, involving the upregulation of the expression of the short heterodimerization partner (SHP-1), which inhibits in turn the activity of several transcription factors. Third, FXR:RXR positively regulates the gene encoding the bile salt export pump (BSEP), which belongs to the ABC transporter superfamily and allows the extrusion of bile acids from hepatocytes into the biliary canaliculus (Ananthanarayanan et al., 2001; Schuetz et al., 2001). These coordinated actions of FXR on CYP7A, NTCP, and BSEP result in lowering the potentially deleterious high levels of bile acids to which hepatocytes are exposed (reviewed in Francis et al., 2003). In contrast, the FXR-mediated induction of the ileal bile acid-binding protein (IBABP), an intra-cellular carrier of bile acids expressed in the ileal epithelial cells, favors the reuptake of bile acids from the gut lumen (Grober et al., 1999). Finally, FXR might also act directly on circulating lipoproteins by inducing the expression and secretion of hepatic ApoCII (Kast et al., 2001), and by increasing the levels of the secreted enzyme phospholipid transfer protein (PLTP) that facilitates the transfer of cholesterol and phospholipids from triglyceride-rich lipoproteins to HDL (Urizar et al., 2000). However, because of the more prominent effects of FXR on bile acid metabolism, it remains uneasy to identify the specific outcome of these increased gene expressions.
In summary, FXR is the transcription factor that senses the intracellular levels of bile acids and is required for limiting bile acid accumulation in the liver. It inhibits bile acid synthesis via the downregulation of CYP7A and increases bile acid efflux in the bile via increased BSEP expression. In the ileal enterocytes, the reabsorbed bile acids are taken in charge by the cytosolic IBABP whose expression is also increased by FXR.
C. AN OVERVIEW OF PPAR:RXR PHYSIOLOGICAL ACTIVITIES
PPARs were the first nuclear receptors identified as "sensors" rather than classic hormone receptors. They are nuclear, lipid-activable molecules that control a variety of genes in several pathways of lipid metabolism (reviewed in Desvergne and Wahli, 1999; Feige et al., 2006). Three isotypes of PPAR, PPARa, PPARb (also called PPARd, NUCI, and FAAR), and PPARg, have been cloned in Xenopus, rodents, and human. Two PPARg isoforms, PPARg1 and PPARg2, are splice variants in their N-terminal domain. PPARa is highly expressed in tissues with high-lipid catabolism, for example liver, brown adipose tissue, skeletal, and heart muscle. PPARb is ubiquitously expressed. PPARg1 is mainly expressed in adipose tissues but is also present in the colon, spleen, retina, hematopoietic cells, and skeletal muscle. PPARg2 has been found mainly in the brown and white adipose tissue (Braissant et al., 1996; Escher et al., 2001). Their modular structure is that of all nuclear receptors. The less conserved N-terminal region bears a ligand-independent activation domain at least in PPARa and PPARg. The DBD is extremely well conserved. The ligand-binding pocket of PPARs is much larger than that of the other nuclear receptors and relatively easily accessible (Xu et al., 2001 and reference therein).
PPARs bind to DNA as heterodimers with RXR on PPAR response elements (PPRE) comprising a direct repeat of two hexamers, closely related to the sequence AGGTCA and separated by one nucleotide (DR-1 sequence). The five nucleotides flanking the 5' end of this core sequence are also important for the efficiency of PPARa:RXR binding.
The first molecules to be recognized as PPARa activators, and later on characterized as ligands, belong to a group of molecules that induce peroxi-some proliferation in rodents, thus explaining the name of PPAR given to this receptor. This diverse group of substances includes, for example, some plasticizers and herbicides. More interestingly, various fatty acids, more particularly unsaturated fatty acids and some eicosanoids mainly derived from arachidonic acid and linoleic acid, bind to PPARa, PPARb, and PPARg with varying affinities. In addition to being activated by fatty acids, PPARa responds to fibrates which are hypolipidemic drugs, and PPARg responds to thiazolidinediones which are insulin sensitizers, demonstrating their potential as drug targets.
In the process of transcriptional regulation, ligand-bound PPARs recruit coactivators, most likely organized in large complexes (Surapureddi et al., 2002). Cofactor recruitment may be PPAR isotype specific and may ensure the specificity of target gene activation. In addition to PPAR ligand binding, PPARs can also be activated by phosphorylation of serines located in the A/B domain (Gelman et al., 2005).
As can be expected from sensors, PPARs, which recognize and bind a variety of fatty acids, regulate in turn most of the pathways linked to lipid metabolism. Most fascinating is their balanced regulatory actions between fatty acid oxidation in the liver and other organs via PPARa, and fatty acid storage in the adipose tissue via PPARg. In contrast, the role of PPARb in metabolism remains elusive, albeit evidence is emerging for its function in lipid and cholesterol metabolism and transport (reviewed in Michalik et al., 2003).
2. PPARg: A Major Regulator of Fatty Acid Storage and Adipogenesis
PPARg is a late marker of adipocyte differentiation, and its artificial expression is sufficient to force fibroblasts to undergo adipogenesis. Whereas PPARg null mice are not viable, due to defects in placenta formation (Barak et al., 1999), the lack of adipocytes carrying the genotype PPARg~'~ in chimeric PPARy+/+:PPARy~/~ mice has demonstrated the importance, in vivo, of PPARg for adipogenesis (Rosen et al., 1999). In addition, mice with an adipose tissue-specific deletion of PPARg exhibit a severe reduction of the number of mature adipocytes both in white and brown adipose tissues, while small and likely nascent adipocytes are appearing. In this model, the expression of the CRE enzyme responsible for the gene deletion is under the activity of the aP2 promoter, thus triggering the deletion only after adipogenesis has taken place. This suggests that PPARg is also essential for the survival of mature adipocytes (He et al., 2003; Imai et al., 2004b). Among PPARg target genes are those encoding the adipocyte fatty acid-binding protein (aP2), the lipoprotein lipase (LPL), the acyl-CoA synthase (ACS), the fatty acid transport protein (FAT/CD36), the glycerol kinase, and the adipose differentiation-related protein (reviewed in Rosen and Spiegelman, 2001). Hence, PPARg target genes are involved in each step of lipid entry and storage in the cells. Finally, PPARg increases the expression of the uncoupling protein, thereby promoting increased energy expenditure via a futile cycle (Guan et al., 2002).
A puzzling observation made a decade ago was that glitazones, which were developed for the treatment of insulin resistance, are PPARg-selective ligands. The link between the promotion of adipocyte differentiation and lipid storage by PPARg and the antidiabetic effects of these compounds is not fully understood. One hypothesis is fat redistribution from muscle to adipose tissue more particularly to subcutaneous fat, which is itself more sensitive to insulin than visceral fat (Gurnell et al., 2003; Wajchenberg, 2000). Alternately, some data support the hypothesis that adiponectin, an adipokine with insulin-sensitizing property and a PPARg target gene, might be a crucial component connecting PPARg activation in the adipose tissue and the metabolic response of the peripheral organs (Gurnell et al., 2003). Other possibilities are the inhibition of hepatic neoglucogenesis or induction of a futile cycle, as mentioned above. Unexpectedly, PPARy+'~ heterozygous mice, rather than being prone to insulin resistance, are partially protected from high-fat diet-induced or monosodiumglutamate-induced weight gain and insulin resistance (Kubota et al., 1999; Miles et al., 2000). A similar protection is obtained via the use of PPARg partial antagonists (Rieusset et al., 2002). Besides the fact that glitazones have nonnegligible side effects, these observations led to the current approaches searching for PPARg modulators rather than for full agonists.
3. PPARb: From Adipogenesis to Fatty Acid Oxidation
The identity of PPARb natural ligands remains the most elusive. Carba-prostacyclin (cPGI), a stable analogue of prostacyclin (PGI2), acts as an agonist of PPARb, supporting the notion that the cyclooxygenase-2 arachi-donate metabolite PGI2 might itself act as a bona fide natural ligand for PPARb (Shao et al., 2002). In addition, like PPARa and PPARg, PPARb binds fatty acids and, therefore, is also most likely a sensor of dietary lipids and lipid derivatives.
PPARb may play a role in the early steps of adipogenesis. Together with two additional transcription factors, C/EBPb and C/EBPd, PPARb appears to be implicated in the induction of PPARg expression (Bastie et al., 1999;
Holst et al., 2003). In turn, high expression of PPARg and C/EBPa in adipocytes establishes and maintains the terminal differentiation program. However, an adipose tissue-specific deletion of PPARb does not alter fat mass (Barak et al., 2002), whereas overexpression of a constitutively active form of PPARb in brown and white adipose tissues generates lean mice and increases the mobilization and oxidation of fatty acids (Wang et al., 2003a). Such activities are also found in the muscle (reviewed in Bedu et al., 2005). In vivo, transgenic mice that overexpress PPARb or a constitutively active PPARb-VP16 fusion protein in muscle exhibit an enrichment of the muscle in red oxidative fibers, with an increased oxidative capacity assessed both at the gene expression and functional levels (Luquet et al., 2003; Wang et al., 2004). Similar results were obtained in wild-type mice treated with the PPARb agonist (GW501516). Such a treatment results in a dose-dependent activation of fatty acid b-oxidation in the muscles, sustained by the higher expression of genes encoding enzymes involved in mitochondrial fatty acid catabolism, such as fatty acid transport proteins (FAT and LCAD) as well as uncoupling protein UCP2 and UCP3, associated to an increased energy expenditure (Tanaka et al., 2003). Interestingly, the energy source of heart muscle cells depends on fatty acid oxidation, and a tissue-specific gene deletion of PPARb in heart led to cardiomyopathy (Cheng et al., 2004). The activity of PPARb also directly affects glucose metabolism, increasing glucose transport in muscle cells (Kramer et al., 2005) and favoring glucose utilization via the pentose phosphate pathway in the liver (Lee et al., 2006). Whereas these results are very encouraging and have prompted the search for PPARb agonists for the treatment of obesity and/or the metabolic syndrome, how and when PPARb is activated in muscle cells, in the liver, or in adipocytes in the physiological context remain to be elucidated. In addition, there is accumulating evidence for an important role of PPARb both in development and in wound healing. The latter involves an active PPARb-dependent prosurvival activity (Di-Poi et al., 2002; Michalik et al., 2001) and raises concerns over the potential but still debated protumorigenic activity of PPARb (reviewed in Michalik et al., 2004).
4. PPARa: A Major Regulator of Fatty Acid Oxidation
PPARa target genes constitute a comprehensive set of genes that participate in many if not all aspects of lipid catabolism. This includes fatty acid transport across the cell membrane (fatty acid transporter protein genes), intracellular binding (liver fatty acid binding protein gene), activation via the formation of acyl-CoA (long-chain fatty acid acyl-CoA synthase gene), catabolism by b-oxidation in peroxisomes and mitochondria, and cata-bolism by «-oxidation in microsomes (acyl-CoA oxidase gene, CYP4A1 and CYP4A6 genes, medium-chain acyl-CoA dehydrogenase gene, and 3-hydroxy-3-methylglutaryl-CoA synthase gene) (reviewed in Desvergne and Wahli, 1999). The role of PPARa in fatty acid oxidation is particularly highlighted during fasting that results in an enhanced load of fatty acids in the liver, then used as the source of energy. Food deprivation provokes an increased expression and activity of PPARa, which stimulates b-oxidation. PPARa null mice, which are viable and exhibit only subtle abnormalities in lipid metabolism when kept under normal laboratory confinement and diet (Lee et al., 1995; Patel et al., 2001), cannot sustain fasting. Their inability to enhance fatty acid oxidation results in hypoketonemia, associated with severe hypothermia and hypoglycemia (Kersten et al., 1999; Leone et al., 1999). Thus, PPARa is crucial for the organism to adapt to an increased demand in fatty acid oxidation, while it seems to play a marginal role in the basal situation with normal diets.
The contribution of PPARa in fatty acid oxidation in muscle tissues, where it is also well expressed, is possibly hidden by other factors, notably PPARb (Muoio et al., 2002). However, high levels of PPARa activity in muscles have been observed in diabetic mice both in heart and skeletal muscle (Finck et al., 2002, 2003; Yechoor et al., 2002). In parallel, overexpression of PPARa in skeletal muscle increases fatty acid oxidation and decreases insulin-stimulated glucose uptake via inhibition of Glut4 expression (Finck et al., 2005), suggesting that the increased activity of PPARa in the muscles of diabetic patients may contribute to insulin resistance. A contrasting pattern is described in the pathological cardiac hypertrophy. In this context, the expression of PPARa is downregulated, the utilization of fatty acids as energy substrate is decreased, and the genes implicated in the utilization of glucose as the main energy source are reinduced (Barger et al., 2000). It is presently unclear whether the decline in PPARa activity and fatty acid oxidation is a cause or a consequence of cardiac hypertrophy. Interestingly, PPARa null mice exhibit a decreased contractile and metabolic reserve in heart, rescued by favoring glucose transport and utilization (Luptak et al., 2005). At present, it is still unclear whether PPARa ligands in human would be beneficial or detrimental to the heart in the context of cardiac hypertrophy.
As a consequence of their activities in lipid metabolism, all three PPARs act on blood lipid levels. PPARa increases reverse cholesterol transport by upre-gulating the expression of the genes encoding the cholesterol acceptor apolipo-protein ApoAI and ApoAII, and that of the hepatic expression of the scavenger receptor BI (SR-BI)/CLA-1, thereby increasing the selective uptake of HDL cholesteryl esters from the blood. Indeed, fibrates have been used for the treatment of dyslipidemia, much before the discovery of their mechanism of action via PPARa. In addition, both PPARa and PPARg upregulate the expression of the lipoprotein lipase gene (reviewed in Bocher et al., 2002). Together with a decreased expression of ApoCIII, these effects increase free fatty acid delivery to peripheral tissues. This explains in part why thiazolidi-nediones, which potently activate PPARg, not only act on insulin sensitivity but also decrease plasma free fatty acid concentrations and improve the overall lipoprotein profile (Mayerson et al., 2002; Oakes et al., 1997).
Treatment with a PPARb agonist of obese rhesus monkeys, used as a relevant animal model for human obesity and the associated metabolic disorders, caused an increase in the level of serum HDL cholesterol, while lowering the level of small-dense LDL, fasting triglycerides, and fasting insulin (Oliver et al., 2001). Similar results were obtained in db/db mice (Leibowitz et al., 2000). Further studies in non-obese mice demonstrate that PPARb activation results in both increased HDL concentration in the blood and accelerated fecal cholesterol removal from the body via downregulation of the intestinal gene expression of cholesterol absorption protein Niemann-Pick C1-like 1 (NPC1L1) (van der Veen et al., 2005).
In summary, each PPAR acts as a lipid sensor with distinct activities that adjust at the cellular and organism levels the metabolic status, with balancing actions on fatty acid oxidation and fatty acid storage processes. These metabolic regulations also involve a PPAR-dependent coordination of glucose metabolism.
IV. THE REXINOID-SIGNALING PATHWAYS: FROM PARTNERSHIP TO LEADERSHIP
As mentioned above, RXR is itself a nuclear receptor that can be activated by 9-cis RA, an isomer of atRA. The present chapter discusses the observations more specifically linked to RXR activities.
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