PPARγ as a metabolic regulator: insights from genomics and pharmacology

PPARγ as a metabolic regulator: insights from genomics and pharmacology David B. Savage Since its identification in the early 1990s, peroxisome-proliferator-activated receptor γ (PPARγ), a nuclear hormone receptor, has attracted tremendous scientific and clinical interest. The role of PPARγ in macronutrient metabolism has received particular attention, for three main reasons: first, it is the target of the thiazolidinediones (TZDs), a novel class of insulin sensitisers widely used to treat type 2 diabetes; second, it plays a central role in adipogenesis; and third, it appears to be primarily involved in regulating lipid metabolism with predominantly secondary effects on carbohydrate metabolism, a notion in keeping with the currently in vogue lipocentric view of diabetes. This review summarises in vitro studies suggesting that PPARγ is a master regulator of adipogenesis, and then considers in vivo findings from use of PPARγ agonists, knockout studies in mice and analysis of human PPARγ mutations/ polymorphisms. Given Western society s propensity for overeating and under-exercising, the major energetic challenge for modern human metabolism is storage of excess calories. This energy overload is predominantly disposed of as triglyceride (TG) in white adipose tissue. The average adipose tissue mass of a 70 kg adult human is 12 kg, representing an energy store of 352 MJ ( 84 000 kcal). By contrast, carbohydrate stores in the form of glycogen in liver and skeletal muscle account for only 6.7 MJ ( 1600 kcal). Unlike hydrophilic glycogen stores, which have a very limited capacity to expand, hydrophobic TG droplets are a much more efficient way to store energy and have considerable potential for expansion. Although this ultimately predisposes over-nourished humans to weight gain, in the short to medium term it represents an efficient metabolic response to fluctuating energy balance. The importance of the capacity to store excess energy in adipocytes is epitomised by the metabolic consequences of a loss of adipose David B. Savage Wellcome Clinician Scientist, Departments of Medicine and Clinical Biochemistry, University of Cambridge, UK. Current address: Yale University School of Medicine, S260 TAC, 1 Gilbert Street, New Haven, CT 06520-8020, USA. Tel: +1 203 737 5679; Fax: +1 203 785 3823; E-mail: david.savage@yale.edu Institute URL: http://www.clbc.cam.ac.uk/ 1

tissue, as seen in lipodystrophy (Ref. 1). The lipodystrophic syndromes encompass a heterogeneous group of conditions characterised by partial or complete absence of adipose tissue due to a lack of functional adipocytes (usually as a result of genetic or immunological mechanisms) (Ref. 2). Lipodystrophy is distinct from leanness, as in that case adipose storage can readily be normalised or even increased above normal by the restoration of a positive energy balance. In lipodystrophic subjects with a significantly reduced adipose tissue storage capacity, positive energy balance is thought to lead to ectopic fat deposition (i.e. lipid deposition in tissues other than fat, such as the liver and skeletal muscle), insulin resistance and ultimately diabetes. Interestingly, obesity is also associated with ectopic lipid accumulation, insulin resistance and diabetes; in this case it is thought that energy intake ultimately exceeds the capacity of adipose tissue to expand, leading to overflow to ectopic sites (Ref. 1). As well as needing to be able to adapt to longterm changes in energy balance, humans have to cope with the energy load associated with consumption of three daily meals. Failure to dispose efficiently of ingested carbohydrate and fat would lead to large changes in plasma glucose and TG. In healthy humans, the liver and skeletal muscle effectively buffer ingested carbohydrate, while adipose tissue plays a key role in buffering postprandial TGs (Ref. 3). Triggered by the discovery of leptin in 1994 (Ref. 4), research over the past decade has yielded significant insight into the complexity of adipocyte differentiation and the mechanisms by which adipose tissue copes with both short- and long-term perturbations in energy balance (Ref. 5). In times of excess, adipose tissue responds by increasing both the number of mature adipocytes by adipogenesis (the differentiation of stromal fibroblast-like preadipocytes into mature lipid-laden adipocytes) and the size of pre-existing cells (hypertrophy). It is also capable of signalling the state of its stores by secreting proteins such as leptin (Ref. 6). Peroxisome-proliferator-activated receptor γ (PPARγ) is one of several transcription factors regulating adipocyte number and size (see Ref. 7 for details of other transcription factors involved in this regulation). It is also involved in modulating secretion of several adipokines (a collective term for proteins secreted by adipose tissue) (Ref. 8) and is therefore uniquely placed to facilitate appropriate storage of excess calories in adipose tissue, to modify signals from adipose tissue to the brain and other tissues regarding the status of these energy stores, and to limit ectopic lipid accumulation in sites such as the liver and skeletal muscle. PPARγ has also been implicated in inflammatory responses and carcinogenesis, but these roles are not covered here for recent comprehensive reviews see Refs 9 and 10. PPARγ PPARγ belongs to the nuclear hormone receptor superfamily. Its name stems from the identification in 1990 of a homologue, PPARα, that mediates the exuberant proliferation of hepatic peroxisomes in mice exposed to various xenobiotic compounds (Ref. 11). When two homologues were later cloned in Xenopus (Ref. 12) and then in all mammalian species studied, the three receptors were designated PPARα, PPARδ and PPARγ. Structure and transcriptional activity There are two protein splice isoforms of PPARγ: PPARγ1 and PPARγ2 (Ref. 13). PPARγ2 has 30 additional N-terminal amino acids compared with PPARγ1 in humans (28 in mice), and is expressed only in adipose tissue. Two additional mrna splice variants PPARγ3 (Ref. 14) and PPARγ4 (Ref. 15) give rise to proteins identical to PPARγ1. The biological relevance of these mrna variants remains unclear. Like many other nuclear receptors, PPARγ contains an N-terminal domain with a ligand-independent AF-1 activation domain, a central DNA-binding domain (DBD) composed of two zinc fingers, and a C-terminal ligand-binding and dimerisation domain (LBD) with a ligand-dependent AF-2 transactivation domain (Fig. 1a). In the absence of ligand, PPARγ can bind to co-repressors (proteins that lead to condensation of chromatin and sequestration of promoter elements), which inhibit transcriptional activity in a DNA-independent manner. All PPARs bind DNA as heterodimers with retinoid X receptor (RXR). Ligand binding to either PPAR or RXR induces conformational changes in the PPAR RXR heterodimer, favouring release of corepressor molecules and recruitment of coactivator proteins, which facilitate access and assembly of a transcriptional regulatory complex (Fig. 1b). This complex binds to specific PPAR 2

a b DNA Co-activator recruitment AF-1 DNA-binding domain Ligand binding PPARγ Ligand-binding domain RXR AGGNCAXAGGNCA PPAR response element PPARγ structure and mode of gene regulation Expert Reviews in Molecular Medicine C 2005 Cambridge University Press PPARγ Co-repressor displacement Gene transcription Figure 1. PPARγ structure and mode of gene regulation. (a) PPARγ (peroxisome-proliferator-activated receptor γ) has the typical nuclear hormone receptor domain structures, including a central DNA-binding domain, a C-terminal ligand-binding domain, and two activation domains (AF-1, ligand-independent activation domain; AF-2, ligand-dependent activation domain). PPARγ1 and 2 are identical except for an additional 30 amino acid N-terminal extension in PPARγ2 (not shown). (b) PPARγ binds to PPAR response elements (PPREs) as an obligate heterodimer with the retinoid X receptor (RXR). Ligand binding to either PPAR or RXR induces displacement of co-repressors, recruitment of co-activators and transcriptional activation of target genes. response elements (PPREs) in the promoter regions of target genes (see Ref. 16 for a detailed review of the mechanism of transcriptional activation). Tissue distribution PPARγ is most highly expressed in both white and brown adipose tissue. The PPARγ2 isoform is almost exclusive to adipose tissue, where it constitutes about 30% of the total PPARγ, whereas PPARγ1 is also readily detectable in large intestine and haematopoietic cells, and is expressed at low levels in liver, skeletal muscle, pancreas and most other tissues (Refs 13, 17). In rodents, PPARγ expression is reduced after an overnight fast and in insulin-deficient streptozotocin-induced diabetes, suggesting that insulin might be involved in stimulating PPARγ expression (Ref. 18). It is of interest to compare the tissue distribution of PPARγ with that of PPARα and of PPARδ for insight into their respective biological roles. PPARα is most highly expressed in muscle (especially in humans) and liver, where it regulates fatty acid oxidation and the metabolic AF-2 response to fasting (Ref. 19). PPARδ is abundantly expressed in almost all tissues. Its precise biological role is less well understood than those of PPARα and PPARγ, but recent work suggests that it is a potent inducer of fatty acid oxidation and that PPARδ agonists improve plasma lipid profiles (Ref. 20). Ligands Whereas membrane-bound receptors are generally responsible for propagating signals initiated by hydrophilic protein molecules, the nuclear hormone receptor superfamily is responsible for signalling by lipophilic molecules, including steroids and derivatives thereof, and fatty acids and their derivatives. Changes in concentration of lipid moieties are sensed by these receptors, which respond by modifying gene transcription in the host cell. The precise nature of the endogenous PPARγ ligand(s) has been the subject of much, as yet unresolved, debate. Naturally occurring fatty acids and eicosanoids can bind and activate all three PPARs. However, most of these naturally occurring ligands bind with relatively 3

low affinity compared with the affinity of wellestablished ligands for other nuclear receptors. They also exist at very low concentrations in vivo (Ref. 21) and are weak agonists, raising doubts about their biological relevance. Tzameli et al. recently identified a higher-affinity lipophilic PPARγ-specific ligand, which was transiently produced in differentiating 3T3L1 pre-adipocytes (Ref. 22). Further work is required to establish the exact nature of this molecule(s) and to determine its relevance to the biological role of PPARγ in mature adipocytes and other sites such as liver and skeletal muscle. Several high-affinity synthetic PPARγ agonists are available. They include thiazolidinediones (TZDs), which were identified as part of a drugscreening process for antidiabetic compounds (Ref. 23) and only later found to act via PPARγ, as well as several more recently identified compounds such as tyrosine-based agonists (Ref. 24). This is an area of intense interest to the pharmaceutical industry, which is pursuing both dual- and pan-ppar agonists (i.e. compounds capable of activating combinations of PPARγ, PPARα and PPARδ) and so-called selective PPARγ modulators (SPPARMs), with both agonist and antagonist activity (see section on SPPARMs below). Target genes The number of genes directly and indirectly regulated by PPARγ continues to expand. The Table 1. Gene targets of PPARγ products of these genes can be grouped functionally into: (1) proteins involved in hydrolysis of plasma TGs, fatty acid uptake and esterification, lipogenesis and TG synthesis; (2) proteins regulating lipolysis; (3) adipokines; and (4) proteins directly implicated in insulin signalling and glucose uptake (Table 1). These targets suggest that PPARγ influences lipid and glucose metabolism within adipocytes (Fig. 2), as well as signalling from adipose tissue. PPARγ agonists also reduce 11β-hydroxysteroid dehydrogenase 1 expression (11β-HSD1) in adipose tissue (Ref. 25). 11β-HSD1 converts inactive cortisone to bioactive corticosterone within tissues, and overexpression of this gene in mouse adipose tissue induces a Cushingoid state in mice, with central obesity, insulin resistance and hypertension (Ref. 26). Hence, suppression of 11β-HSD1 by PPARγ agonists might contribute to their insulinsensitising properties. Key in vitro observations: PPARγ and adipogenesis PPARγ is abundantly expressed in adipocytes and its expression is markedly induced during adipocyte differentiation (Ref. 27), prompting questions about its role in adipogenesis and in mature adipocytes. Overexpression of PPARγ in pre-adipocytes is sufficient for adipogenesis, even in the absence of CCAAT/enhancer-binding protein alpha (C/EBPα), another key transcriptional regulator of adipogenesis (Ref. 28). By contrast, murine embryo fibroblasts (MEFs) lacking PPARγ Function Examples Ref. Triglyceride hydrolysis Lipoprotein lipase 16 Fatty acid uptake/esterification CD36, fatty-acid-transport protein 1 16 Fatty-acid-binding protein 4 (ap2) AcylCoA synthase Lipogenesis and triglyceride synthesis Phosphoenolpyruvate carboxykinase 87 Glycerol kinase 88 Lipolysis regulation Perilipin 89 Adipokines Adiponectin 70 Resistin 90 Insulin signalling and glucose uptake Cbl-associated protein 68 Insulin receptor substrate 2 65 Glucose transporter 4 66 4

Plasma TG LPL NEFA Glycerol CD36 FATP Aquaporin NEFA Glycerol Adipocyte ACS AcylCoA PPARγ target genes and adipocyte metabolism NEFA Figure 2. PPARγ target genes and adipocyte metabolism. PPARγ (peroxisome-proliferator-activated receptor γ) regulates adipocyte metabolism through effects on the transcription of several genes. In the figure, PPARγ-regulated gene products promoting lipid storage are highlighted in green, whereas those promoting lipolysis are highlighted in red (see also Table 1). Plasma triglyceride (TG) is hydrolysed by lipoprotein lipase (LPL) to nonesterified fatty acid (NEFA) and glycerol. NEFA uptake by adipocytes is probably aided by the transporters CD36 and fatty-acid-transport protein (FATP); the aquaporin channel facilitates glycerol transport. In adipocytes, NEFAs are re-esterified via the action of acylcoa synthase (ACS) for storage as TG, while glycerol is converted to glycerol 3-phosphate (G3P) by the action of glycerol kinase (GK). In addition, G3P can be synthesised via the action of phosphoenolpyruvate carboxykinase (PEPCK) (glyceroneogenesis) (dashed lines imply several intermediate steps; PEP, phosphoenolpyruvate). Thus, PPARγ may promote TG synthesis by inducing transcription of genes involved in regulating plasma lipid uptake (LPL, CD36, FATP, aquaporin) and metabolism within the adipocyte (ACS, GK, PEPCK). [Note, however, that GK does not appear to be induced by thiazolidinediones in humans (Ref. 61)]. PPARγ can also influence lipolysis by inducing perilipin expression [perilipin is an important determinant of hormone-sensitive lipase (HSL) activity]. GK Glucose Expert Reviews in Molecular Medicine C 2005 Cambridge University Press TG G3P PEP Pyruvate GLUT4 PEPCK HSL/ perilipin Glycerol are unable to differentiate into adipocytes even in the presence of overexpressed C/EBPα, suggesting that PPARγ is the master regulator of adipocyte differentiation (Ref. 29). In order to examine the relative importance of PPARγ1 and PPARγ2 in adipogenesis, retroviruses were used to restore expression of either PPARγ1 or PPARγ2 to PPARγ-null pre-adipocytes (Ref. 30). Although both proteins were expressed at comparable levels, only PPARγ2 was able to rescue the adipogenic phenotype, suggesting that PPARγ2 is the most important isoform in adipogenesis. Another group obtained different results using a similar experimental paradigm (Ref. 31); their data suggested that adipogenic capacity could be restored to PPARγ-null preadipocytes by overexpressing either PPARγ1 or PPARγ2. However, they also found that the proadipogenic activity of PPARγ2 was greater than that of PPARγ1. This notion is supported by the phenotype of PPARγ2-isoform-specific knockout mice, which have a form of partial lipodystrophy 5

(Ref. 32). Pre-adipocytes isolated from adipose tissue of these mice also fail to differentiate in culture. Animal in vivo observations Rodents have been used to explore the metabolic response to PPARγ agonists in vivo, and to assess the consequences of altering PPARγ expression in both the whole organism and in a tissue-specific manner. Use of PPARγ agonists in rodents Intriguingly, TZDs tend to increase fat mass as well as improving insulin sensitivity and glucose tolerance in rodents and humans. The conventional explanation for these seemingly discordant effects is that TZDs promote lipid uptake and storage in adipose tissue [ fatty acid steal hypothesis (Ref. 33)], thereby lowering systemic free fatty acid (FFA) levels and reducing FFA delivery to other tissues where they have been implicated in inducing insulin resistance (Ref. 34) (Fig. 3). In fact, detailed studies of FFA and TG metabolism indicate that the mode of action of TZDs is more complex than a simple lowering of plasma FFAs. Lipid turnover was initially studied in obese insulin-resistant Zucker rats (fa/fa) treated with TZDs for three weeks (Ref. 35). Rather than a global reduction in plasma FFAs, the authors observed reduced plasma FFA levels in TZD-treated rats exposed to hyperinsulinaemia approximating postprandial levels, and, surprisingly, elevated FFA rates of appearance in the basal (fasting) state of treated animals. The increase in fasting FFA rates of appearance was not, however, associated with elevated plasma FFAs because of a corresponding increase in FFA clearance. Basal FFA oxidation was substantially increased (approximately 50%), while insulin-mediated suppression of fatty acid oxidation was greatly enhanced. Plasma TG levels were also significantly reduced by TZDs, primarily as a result of accelerated conversion of TG-rich very-low-density lipoprotein (VLDL) to TG-poor lipoprotein remnants. These changes in TG and FFA flux are associated with changes in adipose tissue morphology; typically, higher numbers of smaller adipocytes are seen in TZDtreated rodents (Refs 36, 37). TG content in liver and muscle was also significantly reduced (Ref. 35). In an attempt to provide a direct demonstration of the lipid steal hypothesis, normal rats were treated with a TZD (rosiglitazone) for a week before insulin sensitivity and fatty acid disposal were assessed during a TG heparin infusion (Ref. 38). As expected, rosiglitazone improved insulin sensitivity and increased fatty acid uptake into adipose tissue (twofold), while reducing fatty acid uptake into liver and muscle by 30 40%. Taken together, these data suggest that PPARγ activation enhances metabolic flexibility by appropriately facilitating disposal of lipids in adipose tissue in the fed state and FFA turnover in the fasting state (i.e. release of FFAs from adipocytes and their subsequent oxidation). This capacity to synchronise lipid metabolism appropriately with nutrient ingestion is a vital element of normal metabolism in which: (1) postprandial insulin release facilitates glucose uptake and oxidation, glycogen synthesis and lipid disposal in adipose tissue; and (2) FFAs are released by adipocytes in the fasting state when lipid oxidation becomes a key energy source. PPARγ-knockout models PPARγ / PPARγ-knockout mice die in utero as a result of placental insufficiency (Ref. 39). Rescue of PPARγ null embryos by aggregation with tetraploid PPARγ +/+ pre-implantation embryos ultimately resulted in a single live-born PPARγ-null mouse that lacked almost all white and brown adipose tissue (Ref. 39). Another group (Ref. 40) circumvented the problem of placental insufficiency by injecting PPARγ-null embryonic stem cells into wild-type blasts. They found that PPARγ-null cells contributed very little if anything to adipose tissue in these mice, supporting the notion that PPARγ is a key determinant of adipose tissue development. PPARγ +/ If PPARγ agonists improve insulin sensitivity and PPARγ is essential for adipogenesis, one might have expected PPARγ +/ heterozygous mice to be either normal or to have a form of partial lipodystrophy and insulin resistance. Instead, they are protected against both high-fat-dietinduced insulin resistance and ageing-associated insulin resistance (Refs 37, 41). This apparent improvement in insulin sensitivity has been attributed to a reduction in adipocyte size and increased leptin expression. Smaller adipocytes are consistently more sensitive to insulin than are larger adipocytes (Refs 42, 43), and, in addition 6

b Lipotoxicity Muscle Adipocyte hyperplasia and hypertrophy Ectopic lipid accumulation Thiazolidinediones and lipotoxicity Expert Reviews in Molecular Medicine C 2005 Cambridge University Press a Normal energy intake Adipose tissue Excess energy intake Figure 3. Thiazolidinediones and lipotoxicity. Under normal circumstances (a), energy that is not immediately utilised is stored as triglyceride in adipose tissue. Ingestion of excess energy (b) results in adipocyte hyperplasia and hypertrophy, but some of the excess energy is diverted to other tissues such as liver and skeletal muscle where it is believed to induce insulin resistance (so-called lipotoxicity ). PPARγ (peroxisome-proliferator-activated receptor γ) agonists (c) increase the number of small, insulin-sensitive adipocytes. This might lead to weight gain but concurrently lowers ectopic lipid accumulation in liver and skeletal muscle, and improves insulin action. Liver c Thiazolidinedione action Small, insulin-sensitive adipocytes Reduced ectopic lipid accumulation to appetite suppression, leptin might have peripheral insulin-sensitising properties that are anti-steatotic (preventing fat accumulation in liver and muscle) (Ref. 44). Treating PPARγ +/ mice with either PPARγ antagonists or RXR antagonists induced lipodystrophy and insulin resistance, suggesting that although partial PPARγ deficiency might be metabolically beneficial, any additional loss of PPARγ function is deleterious (Ref. 45). Tissue-specific knockouts PPARγ has been knocked out in each of the key insulin-sensitive tissues in mice. Fat-specific PPARγ-knockout mice (generated by crossing PPARγ LoxP mice with ap2-cre mice) manifest progressive lipodystrophy (Ref. 46). They are also more susceptible to high-fat-diet-induced insulin resistance (particularly hepatic insulin resistance), dyslipidaemia and hepatic steatosis than wild-type littermates. Interestingly, although TZD therapy failed to lower FFAs in fatspecific PPARγ-knockout mice, it improved hepatic insulin sensitivity, suggesting that this effect of TZDs is, at least in part, independent of PPARγ activity in adipose tissue. The relative importance of PPARγ in adipose tissue in mediating the metabolic response to TZDs has also been examined by treating mice without 7

any adipose tissue (i.e. lipodystrophic mice) with TZDs (Refs 47, 48). The response is somewhat variable depending upon the extent of the lipodystrophy and the background rodent strain, but in general it is less dramatic than that seen in mice with adipose tissue, emphasising the importance of adipose tissue in TZD action; however, the fact that lipodystrophic mice can respond does suggest that PPARγ might also be biologically active in other tissues. Muscle-specific PPARγ-knockouts have been generated by two independent groups. According to Hevener et al. (Ref. 49), muscle-specific PPARγknockout mice weigh more than wild-type mice, and manifest insulin resistance (whole-body and muscle), dyslipidaemia and fatty liver. TZDs improve whole-body insulin sensitivity, dyslipidaemia and fatty liver in these mice but fail to improve muscle insulin sensitivity. Norris et al. (Ref. 50) also detected obesity and wholebody insulin resistance in muscle-specific PPARγknockout mice but, unlike Hevener et al. (Ref. 49), they suggested that muscle insulin sensitivity was within normal limits. They also noted that the mutant mice responded to TZDs as well as wild-type mice, seemingly suggesting that PPARγ expression in muscle was not required for TZD action. The differences in muscle insulin sensitivity between these studies are important as both studies set out to determine the biological relevance of PPARγ expression in muscle. Mice lacking PPARγ in the liver have an increase in fat mass, dyslipidaemia and insulin resistance (Refs 51, 52). Crossing these mice with lipodystrophic AZIP mice (Ref. 51) or leptin-deficient ob/ob mice (Ref. 52) lowers liver TGs but worsens muscle insulin resistance and dyslipidaemia, suggesting that PPARγ in the liver might be involved in limiting plasma hypertriglyceridaemia and excess lipid delivery to skeletal muscle. This might, however, come at the cost of hepatic steatosis, at least in circumstances in which the excess lipids cannot be diverted to adipose tissue. In nonlipodystrophic mice, loss of hepatic PPARγ does not alter the response to TZD treatment, suggesting that liver PPARγ is not essential for TZD action (Ref. 51). Targeted elimination of PPARγ in pancreatic β cells does not alter glucose homeostasis but does affect β-cell proliferation (Ref. 53). This is in keeping with the regulatory role of PPARγ in adipocyte differentiation and carcinogenesis (Ref. 10). Inflammation in adipose tissue One further possible mechanism by which PPARγ could alter metabolism involves the recently described inflammatory response within adipose tissue in obese rodents and humans. Macrophages constitute a significant proportion of the stromovascular fraction of adipose tissue and their numbers are significantly increased in obese states, where they appear to make a substantial contribution to gene expression within adipose tissue (Refs 54, 55). Whether this inflammatory infiltrate is responsible for the development of insulin resistance in obese states is not yet clear, although Xu et al. (Ref. 54) did suggest that the increase in inflammatory gene expression within adipose tissue preceded the dramatic increase in plasma insulin levels noted in high-fat-fed mice. They also reported downregulation of these macrophage-derived genes in response to treatment with TZDs. This observation is in keeping with the reported increase in inflammation within adipose tissue of adipocyte-specific PPARγ-knockout mice (Ref. 46). Although much work remains to be done in this area, PPARγ is expressed at significant levels in macrophages (Ref. 56) and it might provide yet another mechanism by which PPARγ affects insulin sensitivity. Human studies Use of PPARγ agonists in humans In insulin-resistant humans, TZDs improve insulin sensitivity and glucose tolerance (Ref. 57). Pioglitazone also lowers TGs and raises highdensity lipoprotein (HDL), whereas rosiglitazone has no effect on fasting TGs (Refs 57, 58, 59). Pioglitazone s capacity to lower TGs is probably a result of weak PPARα agonist activity (Ref. 60). Although TZDs are also frequently said to lower FFAs (Ref. 57), this observation is not universally accepted (Refs 59, 61) and might be a little simplistic. Recently reported studies in humans suggest that whereas rosiglitazone does not lower fasting FFAs and TGs in type 2 diabetics, it does lower postprandial FFAs and TGs (Ref. 59). The capacity to improve insulin sensitivity also improves ovarian function in women with polycystic ovary syndrome (Ref. 62). However, there is a cost to pay for the metabolic benefits namely weight gain. In fact, the extent of metabolic improvement is often proportional to weight gain. Fluid retention (an ill-understood side effect of TZDs), reduced urinary caloric losses 8

in the form of glucosuria in diabetics, and an increase in fat mass all contribute to the weight gain. The increase in fat mass is also associated with fat redistribution between adipose tissue depots [from visceral to subcutaneous depots (Ref. 57), and from the liver, and arguably muscle, to adipose tissue (Refs 63, 64)]. Insulin sensitivity is consequently improved in both liver and skeletal muscle (Ref. 63). Taken together, these observations suggest that the key component in the insulin-sensitising activity of PPARγ agonists is postprandial lipid trapping and storage in adipose tissue, with secondary improvements in insulin-stimulated suppression of hepatic glucose output and glucose disposal in skeletal muscle. However, TZDs might also have direct effects on insulin signalling intermediates. Increases in expression of insulin receptor substrate 2 (IRS2) (Ref. 65) and the glucose transporter GLUT4 (Ref. 66) have been reported in subjects treated with TZDs, suggesting that, in addition to indirect consequences of changes in lipid metabolism, PPARγ might also have direct effects on glucose metabolism. Saltiel and others have recently described a so-called second signalling pathway by which insulin receptor phosphorylation ultimately induces GLUT4 translocation to the plasma membrane and subsequent glucose transport (Ref. 67). In addition to the well-established pathway involving IRS1/2 and phosphoinositol 3-kinase (PI3K), they described a pathway involving Cbl-associated protein (CAP), c-cbl, Cbl-b, CrkII and the TC10 GTPase. The CAP gene has a PPRE in its promoter and is induced by TZDs, potentially explaining the capacity of TZDs to increase insulin-stimulated glucose uptake (Ref. 68). However, Mitra et al. (Ref. 69) recently knocked out CAP, c-cbl plus Cbl-b, or CrkII using RNA interference in cell culture without altering insulin-stimulated glucose uptake, raising concerns about the biological relevance of this pathway in insulin-induced GLUT4 translocation to the plasma membrane. TZDs might also modify insulin action in humans by regulating secretion of adipokines from adipose tissue (Ref. 57). Although the capacity of adipose tissue to secrete proteins with endocrine activity is no longer in doubt (leptin being the best-characterised example), many of the other proteins encompassed by the term adipokine are either predominantly produced by stromovascular cell types within adipose tissue and/or other tissues such as the liver, a fact that makes it difficult to discern the biological importance of the fraction of these proteins produced by adipose tissue. Adipokines probably have a role in altering insulin action in disease states such as sepsis, but their role outside such states remains unproven in humans. Perhaps the most exciting candidate in this class of proteins is adiponectin, which is almost exclusively produced by adipocytes and whose plasma levels are regulated by TZDs (Refs 70, 71; see Ref. 72 for review). Human genetic variants N-terminal mutations/polymorphisms Several mutations have been identified in the PPARG gene, providing novel insights into the biological role of PPARγ in humans (Fig. 4). By far the most common variant is specific to PPARγ2 and results in a proline to alanine substitution at position 12 (Pro12Ala) (minor allele frequency is about 12% in Caucasians). Carriers of the Ala variant, which has less transcriptional activity than the Pro form, were originally reported to be leaner than Pro carriers and to be protected against diabetes (Ref. 73). Although subsequent studies failed to verify the initial observation, a metaanalysis undertaken by Altshuler et al. (Ref. 74) reported a modest (1.25-fold) but statistically significant (P = 0.002) increase in diabetes risk in Pro carriers. Given the prevalence of the Pro12Ala variant, it is arguably currently the dominant genetic variant associated with diabetes. The inconsistent association studies of this variant probably reflects the importance of gene gene and gene environment interactions in determining the ultimate human phenotype. This notion is supported by a report indicating that variations in dietary polyunsaturated fat versus saturated fat intake influence body mass index in Ala carriers (Ref. 75). A much rarer, gain-of-function mutation in PPARγ Pro115Gln was identified in four morbidly obese people (Ref. 76). This mutation prevents phosphorylation of the serine residue at postion 114 and enhances transcription of PPARγ target genes. Although, it was suggested that Pro115Gln carriers had relatively little impairment in insulin sensitivity despite their obesity, they were diabetic and formal measurements of insulin sensitivity were not undertaken. Mice were recently generated with a homozygous Ser112Ala PPARγ mutation in an effort to further explore the 9

impact of N-terminal PPARγ phosphorylation (Ref. 77). This mutation is similar to the Pro115Gln mutation as it prevents phosphorylation of Ser112 (equivalent to Ser114 in humans) and enhances transcriptional activity. Surprisingly, in contrast to the human phenotype, these mice were not obese and were protected against insulin resistance in the setting of diet-induced obesity. (Discrepancies between humans and analogous rodent models are discussed further below.) Ligand-binding-domain mutations More recently, several heterozygous loss-offunction mutations have been identified within the LBD of PPARγ (Ref. 78) (Fig. 4). In vitro characterisation of these mutations suggested that: (1) the ability of the mutant proteins to bind pharmacological PPARγ agonists was significantly impaired; (2) the transcriptional activity of the mutants was significantly reduced; and (3) when co-expressed with equal amounts Pro12Ala Pro115Gln FS of the wild-type receptor, the mutants inhibited wild-type transcriptional activity that is, they exhibited dominant negative behaviour (Ref. 79). To date, all adult carriers of dominant negative LBD mutations have a stereotyped form of partial lipodystrophy with selective loss of limb and gluteal fat. Facial fat is variably preserved, in contrast to the increase in facial fat noted in subjects with familial partial lipodystrophy (FPLD) due to LMNA (lamin A/C) mutations. The lipodystrophy appears to be milder than that seen in typical FPLD, and is particularly difficult to discern in men. All affected subjects have severe insulin resistance, and two children, aged 3 and 7 years, with the Pro467Leu mutation were also hyperinsulinaemic, suggesting that insulin resistance is a very early feature of this condition (Ref. 80). Additional features include a propensity to develop early-onset diabetes, dyslipidaemia, fatty liver and hypertension. Acanthosis nigricans and features of hyperandrogenism (polycystic Val290Met Dominant loss of function Partial lipodystrophy Dyslipidaemia Insulin resistance Phe360Leu Arg397Cys Pro467Leu PPARγ1/2 Gain of function Morbid obesity Loss of function? Increased susceptibility to diabetes Loss of function? Lower BMI and reduced diabetes risk DNA-binding domain Ligand-binding domain Human PPAR γ variants Expert Reviews in Molecular Medicine C 2005 Cambridge University Press Figure 4. Human PPARγ variants. Human genetic variants identified thus far, and their principal phenotypic features, are indicated on a schematic representation of the domain structure of PPARγ (peroxisomeproliferator-activated receptor γ). Dashed lines represent the PPARγ2-specific N-terminus. BMI, body mass index; FS, (A553 AAAiT)fs.185(stop 186). 10

ovary syndrome) were also seen in several carriers. Although adipose tissue morphology was normal in the only subject in whom this was examined, adipose tissue function was clearly disturbed in the same individual (Ref. 80). The usual postprandial increase in plasma TG clearance across subcutaneous abdominal adipose tissue was not seen; in fact, the clearance was very low both in the fasting state and postprandially. Interestingly, adipose tissue lipolysis, as assessed by glycerol output, was also low both in the fasted and postprandial states (Ref. 80). In addition to partial lipodystrophy, abnormal adipose tissue function and fatty liver, carriers of LBD mutations have very low plasma adiponectin levels (Ref. 80), providing yet another potential mechanism for the observed insulin resistance. Insulin resistance is frequently associated with hypertension, but the early onset and severity of hypertension seen in some carriers of PPARγ LBD mutations suggests that PPARγ might have additional effects on blood pressure regulation (Ref. 79). This notion is of particular interest as the principal phenotypic abnormality noted in mice harbouring the rodent equivalent of the Pro467Leu mutation was hypertension (Ref. 81). These mice had partial lipodystrophy but apparently normal insulin sensitivity. Although the reasons for the striking differences between human and rodent phenotypes of both the gain-of-function N-terminal and lossof-function LBD mutants remain ill understood, these observations emphasise the need for discretion when translating insights from rodent metabolism into humans. DNA-binding-domain mutation The PPARG frameshift (FS) mutant, which results in a premature stop mutation within the DNAbinding domain (Fig. 4), differs from all the LBD mutants in several respects (Ref. 82). First, it does not exhibit in vitro dominant negative activity when co-transfected with wild-type PPARγ. Second, although one of the female carriers does appear to have partial lipodystrophy, and fat mass is lower than that predicted by height and weight in all carriers, partial lipodystrophy is not clinically apparent in several carriers of the mutation. Third, two male carriers of the mutation had normal fasting insulin concentrations and all five insulin-resistant carriers were also heterozygous for a second FS premature stop mutation in the muscle-specific regulatory subunit of phosphoprotein phosphatase 1 (PPP1R3A) (Ref. 82). PPP1R3A is a key regulator of glycogen metabolism in skeletal muscle. Although the human PPARG FS mutant is not identical to the rodent PPARγ +/ model, both phenotypes manifest normal insulin sensitivity on regular diets and yet render carriers susceptible to additional metabolic insults [in the form of PPARγ or RXR antagonists in mice (Ref. 45) and a second mutation in humans (Ref. 82)]. The ability of mutations that in isolation produce a mild phenotype, to induce severe phenotypic states in the presence of a second hit, whether it be environmental or genetic, might represent the sort of interactions responsible for complex conditions such as type 2 diabetes. SPPARMs and dual-/pan-ppar agonists The fact that PPARγ agonists appear to have two distinct metabolic effects promotion of adipogenesis and improved insulin sensitivity offers the potential for these two effects to be independently regulated. Rocchi et al. (Ref. 83) identified a PPARγ ligand, N-(9-fluorenylmethyloxycarbonyl) (FMOC)-L-leucine, capable of improving insulin sensitivity without promoting adipogenesis. This capacity to modulate nuclear receptor activity selectively has already been exploited in the oestrogen receptor field, where selective oestrogen receptor modulators (SERMs) such as tamoxifen and raloxifene behave as anti-oestrogens in breast tissue, and as agonists of the oestrogen receptor in bone (Ref. 84). The tissue-specific effects of SERMs probably result from selective ligandinduced interactions between the oestrogen receptor and cofactors (co-activators and corepressors) (Ref. 85). When FMOC-L-leucine binds to PPARγ it induces recruitment of a different set of co-activators to those recruited in the presence of TZDs, suggesting that selective PPAR modulators (SPPARMs) might also be a viable therapeutic option. The other approach being utilised in efforts aiming to separate the improvements in insulin sensitivity from increased adipogenesis is the development of dual- and pan-ppar agonists. As PPARα and PPARδ agonists promote fat oxidation, the hope is that dual- and/or pan-ppar agonists might improve insulin sensitivity while promoting weight loss rather than weight gain (Ref. 86). Conclusions PPARγ is most highly expressed in adipose tissue where it is essential for adipogenesis. In this 11

regard, PPARγ2 appears to be more important than PPARγ1. PPARγ also plays a key role in coordinating postprandial lipid uptake into adipocytes and release of free fatty acids in the fasting state for utilisation by other oxidative tissues such as liver and skeletal muscle. Failure of the capacity to store excess energy in adipose tissue and/or failure to synchronise lipid trafficking into adipose tissue with ingestion of food results in ectopic lipid accumulation in liver and skeletal muscle, and insulin resistance. The recently acknowledged capacity of adipocytes to signal the status of energy stores to other tissues such as the brain (via leptin), liver and skeletal muscle (possibly via adiponectin) is also subject to regulation by PPARγ. In my view, the available evidence strongly favours a predominant metabolic role for PPARγ within adipose tissue; however, tissue-selective rodent knockout models do suggest that low level expression of PPARγ within liver and skeletal muscle might also be biologically relevant. Acknowledgements and funding D.B.S. is supported by The Wellcome Trust, UK. The author thanks the anonymous peer reviewers for their comments. References 1 Friedman, J. (2002) Fat in all the wrong places. Nature 415, 268-269, PubMed: 11796987 2 Garg, A. (2004) Acquired and inherited lipodystrophies. N Engl J Med 350, 1220-1234, PubMed: 15028826 3 Frayn, K.N. (2002) Adipose tissue as a buffer for daily lipid flux. Diabetologia 45, 1201-1210, PubMed: 12242452 4 Zhang, Y. et al. (1994) Positional cloning of the mouse obese gene and its human homologue. Nature 372, 425-432, PubMed: 7984236 5 Spiegelman, B.M. and Flier, J.S. (2001) Obesity and the regulation of energy balance. Cell 104, 531-543, PubMed: 11239410 6 Kershaw, E.E. and Flier, J.S. (2004) Adipose tissue as an endocrine organ. J Clin Endocrinol Metab 89, 2548-2556, PubMed: 15181022 7 Rosen, E.D. et al. (2000) Transcriptional regulation of adipogenesis. Genes Dev 14, 1293-1307, PubMed: 10837022 8 Rangwala, S.M. and Lazar, M.A. (2004) Peroxisome proliferator-activated receptor gamma in diabetes and metabolism. Trends Pharmacol Sci 25, 331-336, PubMed: 15165749 9 Daynes, R.A. and Jones, D.C. (2002) Emerging roles of PPARs in inflammation and immunity. Nat Rev Immunol 2, 748-759, PubMed: 12360213 10 Michalik, L., Desvergne, B. and Wahli, W. (2004) Peroxisome-proliferator-activated receptors and cancers: complex stories. Nat Rev Cancer 4, 61-70, PubMed: 14708026 11 Issemann, I. and Green, S. (1990) Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. Nature 347, 645-650, PubMed: 2129546 12 Dreyer, C. et al. (1992) Control of the peroxisomal beta-oxidation pathway by a novel family of nuclear hormone receptors. Cell 68, 879-887, PubMed: 1312391 13 Tontonoz, P. et al. (1994) mppar gamma 2: tissue-specific regulator of an adipocyte enhancer. Genes Dev 8, 1224-1234, PubMed: 7926726 14 Fajas, L., Fruchart, J.C. and Auwerx, J. (1998) PPARgamma3 mrna: a distinct PPARgamma mrna subtype transcribed from an independent promoter. FEBS Lett 438, 55-60, PubMed: 9821958 15 Sundvold, H. and Lien, S. (2001) Identification of a novel peroxisome proliferator-activated receptor (PPAR) gamma promoter in man and transactivation by the nuclear receptor RORalpha1. Biochem Biophys Res Commun 287, 383-390, PubMed: 11554739 16 Desvergne, B. and Wahli, W. (1999) Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocr Rev 20, 649-688, PubMed: 10529898 17 Escher, P. et al. (2001) Rat PPARs: quantitative analysis in adult rat tissues and regulation in fasting and refeeding. Endocrinology 142, 4195-4202, PubMed: 11564675 18 Vidal-Puig, A. et al. (1996) Regulation of PPAR gamma gene expression by nutrition and obesity in rodents. J Clin Invest 97, 2553-2561, PubMed: 8647948 19 Kersten, S., Desvergne, B. and Wahli, W. (2000) Roles of PPARs in health and disease. Nature 405, 421-424, PubMed: 10839530 20 Evans, R.M., Barish, G.D. and Wang, Y.X. (2004) PPARs and the complex journey to obesity. Nat Med 10, 355-361, PubMed: 15057233 21 Bell-Parikh, L.C. et al. (2003) Biosynthesis of 15- deoxy-delta12,14-pgj2 and the ligation of PPARgamma. J Clin Invest 112, 945-955, PubMed: 12975479 22 Tzameli, I. et al. (2004) Regulated production of a peroxisome proliferator-activated receptor- 12

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Features associated with this article Figures Figure 1. PPARγ structure and mode of gene regulation. Figure 2. PPARγ target genes and adipocyte metabolism. Figure 3. Thiazolidinediones and lipotoxicity. Figure 4. Human PPARγ variants. Table Table 1. Gene targets of PPARγ. Further reading, resources and contacts Saltiel, A.R. and Kahn, C.R. (2001) Insulin signalling and the regulation of glucose and lipid metabolism. Nature 414, 799-806, PubMed: 11742412 Shulman, G.I. (2000) Cellular mechanisms of insulin resistance. J Clin Invest 106, 171-176, PubMed: 10903330 Stumvoll, M. and Haring, H. (2002) The peroxisome proliferator-activated receptor-gamma2 Pro12Ala polymorphism. Diabetes 51, 2341-2347, PubMed: 12145143 Yki-Jarvinen, H. (2004) Thiazolidinediones. N Engl J Med 351, 1106-1118, PubMed: 15356308 Recent related article in Expert Reviews in Molecular Medicine: Clarke, N. et al. (2004) Retinoids: potential in cancer prevention and therapy. Expert Rev Mol Med 6, 1-23, PubMed: 15569396 Citation details for this article David B. Savage (2005). Expert Rev. Mol. Med. Vol. 7, Issue 1, 24 January, DOI: 10.1017/S1462399405008793 16